[Growth Hormone (GH) Receptor] Antibody

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Molecular Endocrinology 18(12):2981–2996 Copyright © 2004 by The Endocrine Society doi: 10.1210/me.2004-0102

A Conformationally Sensitive GHR [Growth Hormone (GH) Receptor] Antibody: Impact on GH Signaling and GHR Proteolysis JING JIANG,* XIANGDONG WANG,* KAI HE, XIN LI, CHANGMIN CHEN, PETER P. SAYESKI, MICHAEL J. WATERS, AND STUART J. FRANK Department of Medicine (J.J., X.W., S.J.F.), Division of Endocrinology, Diabetes, and Metabolism, Department of Cell Biology (K.H., S.J.F.), Department of Pathology (X.L.), University of Alabama at Birmingham, Birmingham, Alabama 35294-0012; School of Biomedical Sciences and Institute for Molecular Bioscience (C.C.), University of Queensland, Brisbane, Queensland, Australia; Department of Physiology and Functional Genomics (P.P.S.), University of Florida College of Medicine, Gainesville, Florida 32610; and Endocrinology Section (S.J.F.), Medical Service, Veterans Affairs Medical Center, Birmingham, Alabama 35233 The GH receptor (GHR) mediates metabolic and somatogenic actions of GH. Its extracellular domain (ECD; residues 1–246) has two subdomains, each with seven ␤ strands organized into two antiparallel ␤ sheets, connected by a short hinge region. Most of the ECD residues involved in GH binding reside in subdomain 1, whereas subdomain 2 harbors a dimerization interface between GHR dimers that alters conformation in response to GH. A regulated GHR metalloprotease cleavage site is in the membrane-proximal stem region of subdomain 2. We have identified a monoclonal antiECD antibody, anti-GHRext-mAb, which recognizes the rabbit and human GHRs by immunoprecipitation, but less so after GH treatment. By immunoblotting and immunoprecipitation, anti-GHRext-mAb recognized a glutathione-S-transferase (GST) fusion incorporating subdomain 2, but not one including subdomain 1. In transient transfection experiments, anti-GHRext-mAb failed to recognize by immunoprecipitation a previously characterized dimerization interface mutant GHR that is incompetent for signaling. In signaling experiments, brief

pretreatment of GH-responsive human fibrosarcoma cells with anti-GHRext-mAb dramatically inhibited GH-induced Janus kinase 2 and signal transducer and activator of transcription 5 tyrosine phosphorylation and prevented GH-induced GHR disulfide linkage (a reflection of GH-induced conformational changes). In contrast, anti-GHRext-mAb only partially inhibited radiolabeled GH binding, suggesting its effects on signaling were not simply via inhibition of binding. Furthermore, anti-GHRext-mAb prevented phorbol ester-stimulated GHR proteolysis, but GHR cleavage site mutants were normally recognized by the antibody, indicating that the stem region cleavage site is not a direct epitope. A Fab fragment of anti-GHRext-mAb inhibited GH-induced GHR disulfide linkage and signaling, as well as phorbol ester-induced GHR proteolysis, in a fashion similar to the intact antibody. Thus, our findings suggest that anti-GHRext-mAb has promise as a GH antagonist and as a tool in studies of conformational changes required for GHR activation. (Molecular Endocrinology 18: 2981–2996, 2004)

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Crystallographic studies indicate that the ECD is divided into two ␤ sandwich subdomains, each comprised of seven ␤ strands organized into two antiparallel ␤ sheets (4). Subdomains 1 and 2 (residues 1–123 and 128–246, respectively) are connected by a fourresidue hinge region. Of the nine GHR residues known to interact in the crystal structure with GH, seven (residues 43, 44, 103, 104, 120, 126, and 127) reside in subdomain 1 and/or the hinge region; only residues 165 and 166 lie within subdomain 2 (4). Additional residues shown by mutagenesis to be relevant in GH binding include residues 105 and 106 in subdomain 1 and 164 and 169 in subdomain 2 (5). GH interacts with the receptor ECD in a 1:2 GH:GHR stoichiometry with two sites on the GH molecule (site 1 and site 2) binding sequentially to the two receptors in the complex (6). The ligand-bound GHRs also interact with each other

HE GH RECEPTOR (GHR) is a glycoprotein member of the type 1 cytokine receptor superfamily that mediates the normal metabolic and somatogenic actions of GH (1–3). The mature protein is 620 amino acids (in human and rabbit) in length, with residues 1–246 constituting the extracellular domain (ECD). * J.J. and X.W. were equal contributors to this work. Abbreviations: anti-GHRext-mAb, Monoclonal anti-ECD antibody; dsl, disulfide-linked; ECD, extracellular domain; Epo, erythropoietin; GHBP, GH binding protein; GHR, GH receptor; GST, glutathione-S-transferase; h, human; JAK, Janus kinase; PMA, phorbol-12-myristate-13-acetate; rbGHR, rabbit GHR; STAT, signal transducer and activator of transcription; WT, wild-type. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.

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at several contact point residues, constituting a dimerization interface. In contrast to the amino acids that participate in interaction with GH, the ECD residues that form the dimerization interface are confined to subdomain 2 of the receptor. Unlike the situation in the presence of GH, very little information exists about the structure of the unliganded GHR, and until recently it was believed that the receptor exists as a monomer before GH exposure. However, Gent et al. (7) used coimmunoprecipitation to demonstrate that GHR dimerization can be observed even in the absence of GH, suggesting that GH induces a conformational change in the receptor dimer, rather than dimerization per se, to cause receptor activation. The nature of this conformational change remains obscure but may involve approximation of dimerization interface residues in subdomain 2 and/or other more distal stem region residues (238– 246) within this subdomain (8). We previously reported initial characterization of a monoclonal anti-GHR antibody, designated antiGHRext-mAb, raised against a bacterially expressed GST fusion protein incorporating the entire ECD of the rabbit (rb) GHR (9). This antibody cross-reacts with the human (h) GHR and displayed conformational sensitivity in its interaction with the receptor. Immunoprecipitation of cellular GHRs was dose-dependently inhibited by GH treatment before extraction of cellular proteins. Treatment with a GH antagonist that binds the receptor via its GH site 1, but fails to activate receptor signaling, did not prevent anti-GHRext-mAb immunoprecipitation; however, the antagonist reversed the inhibitory effect of GH on anti-GHRext-mAb immunoprecipitation, suggesting that anti-GHRext-mAb was not simply competing for the GH binding sites on the receptor (or at least the GHR sites involved in interaction with GH site 1). Thus, loss of anti-GHRext-mAb immunoprecipitability correlated with GH-induced attainment of a GHR conformation competent for signaling and could be used along with GH-induced GHR disulfide linkage as biochemical markers of conformational changes in the receptor (9, 10). In this report, we further characterized anti-GHRext-mAb, determining that its epitope(s) is contained with subdomain 2, but not subdomain 1, of the rbGHR and that it can interact with the receptor on the surface of intact cells. We also tested the effects of anti-GHRext-mAb on GHR signaling and on regulated GHR proteolysis. Our results suggest that this antibody’s conformational sensitivity rests with its ability to interact with an intact dimerization interface within the receptor and emphasize the importance of that receptor region for attainment of the proper signaling conformation. Our results also suggest that regulated GHR proteolysis, which is mediated by metalloprotease activity and alters cellular sensitivity to GH (11–15), is influenced by antiGHRext-mAb binding, even though the proteolytic site is not an epitope for the antibody. Furthermore, a Fab fragment of anti-GHRext-mAb retains these characteristics regarding GHR signaling and proteolysis. Col-

Jiang et al. • Conformationally Sensitive GHR Antibody

lectively, these data indicate that anti-GHRext-mAb has promise as a GH antagonist and as a tool in studies of GH-induced receptor conformational changes.

RESULTS Anti-GHRext-mAb Recognizes Cell Surface GHRs via Subdomain 2 and Is Sensitive to GH-Induced Conformational Change in the Receptor Anti-GHRext-mAb is a monoclonal antibody raised against the rbGHR ECD that cross-reacts with the human and bovine GHRs (Refs. 9 and 12 and data not shown). We have recently developed by stable transfection a human fibrosarcoma cell line, ␥2A-rbGHR/ Janus kinase (JAK) 2 (herein referred to as C14), that expresses rbGHR and responds to GH with GHR, JAK2, and signal transducer and activator of transcription (STAT) 5 tyrosine phosphorylation and activation (16). We initially used this cell line to test the capacity of anti-GHRext-mAb to recognize the cell surface GHR by immunoprecipitation in two types of experiments (Fig. 1). First, serum-starved cells were treated with (⫹) or without (⫺) GH for 15 min at 37 C, after which they were washed and detergent solubilized. Lysates were immunoprecipitated with either anti-GHRext-mAb or anti-GHRcyt-mAb (Fig. 1A, lanes 1 and 2 and 3 and 4, respectively) and eluted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. Anti-GHRcyt-mAb is an independently derived monoclonal antibody directed at the hGHR cytoplasmic domain that cross-reacts with rbGHR (9, 15); antiGHRcyt-AL47 is a rabbit serum also raised against the hGHR cytoplasmic domain that reacts with GHRs from several species (14, 15, 17, 18). As previously observed in other cell types (9), treatment of C14 cells with GH resulted in a loss of immunoprecipitation of GHR by anti-GHRext-mAb (lane 2 vs. 1), but not by anti-GHRcyt-mab (lane 4 vs. 3). We next tested the ability of anti-GHRext-mAb to recognize GHRs on the surface of intact cells by assessing surface immunoreactivity. In the experiment shown in Fig. 1B, serum-starved C14 cells were incubated with either anti-GHRext-mAb (lane 1) or, as a control, anti-GHRcyt-mAb (lane 2) for 1 h at 4 C. After washing off unbound antibodies, cells were detergentsolubilized and protein G-Sepharose was added to the lysate to precipitate the antibodies. Immunoblotting of the resolved eluates with anti-GHRcyt-AL47 revealed that anti-GHRext-mAb, but not anti-GHRcyt-mAb, recognized GHRs in this assay (lane 1 vs. 2). To determine the effect of GH treatment on the ability of antiGHRext-mAb to react with the surface GHR, we first performed the experiment shown in Fig. 1C. Serumstarved C14 cells were treated with (⫹) or without (⫺) GH for 15 min at 37 C, after which the cells were washed and reincubated with either anti-GHRext-mAb (lanes 1 and 2) for 60 min at 4 C. After washing off


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Fig. 1. Anti-GHRext-mAb Conformation Specifically Recognizes the Surface GHR A, Standard immunoprecipitation. Serum-starved C14 cells were treated with (⫹) or without (⫺) GH (500 ng/ml) for 15 min, as indicated. Cells were detergent solubilized and extracts were immunoprecipitated with either anti-GHRext-mAb (lanes 1 and 2) or anti-GHRcyt-mAb (lanes 3 and 4). Eluates of each precipitate were resolved by SDS-PAGE and GHRs were detected by immunoblotting with anti-GHRcyt-AL47. The position of the 119-kDa molecular mass marker is indicated, as is the position of mature GHR. The data shown are representative of two such experiments. B, Specificity of surface immunoreaction. Anti-GHRext-mAb (lane 1) or anti-GHRcyt-mAb (lane 2) were added to the serum-starved C14 cells for 1 h at 4 C, as in Materials and Methods, before washing and detergent solubilization; protein G-Sepharose was added to the extracts to precipitate surfacebound antibodies. Eluates of each precipitate were resolved by SDS-PAGE and GHRs were detected by immunoblotting with anti-GHRcyt-AL47. The position of the 119-kDa molecular mass marker is indicated, as is the position of mature GHR (bracket). The data shown are representative of three such experiments. C and D, Effect of GH treatment on surface immunoreaction. Serum-starved C14 cells were treated with (⫹) or without (⫺) GH (500 ng/ml) for 15 min at 37 C (C) or 90 min at 4 C (D), followed by addition of anti-GHRext-mAb for 45 min (D, lanes 1 and 2) or 60 min (C, lanes 1 and 2) at 4 C, as in Materials and Methods. After washing and detergent solubilization, protein G-Sepharose was added to the extracts to precipitate surface-bound antibodies. Eluates of each precipitate (C and D, lanes 1 and 2) and aliquots of unprecipitated extracts (C and D, lanes 3 and 4) were resolved by reduced SDS-PAGE and GHRs were detected by immunoblotting with anti-GHRcyt-AL47. In D, aliquots of extract were resolved by nonreduced SDS-PAGE before immunoblotting (lanes 5 and 6). The position of the 119-kDa molecular mass marker is indicated, as is the position of mature GHR (bracket or arrow) and immature GHR (arrowhead). In the nonreduced immunoblot (D, lanes 5 and 6), the dsl and non-dsl forms of the GHR are noted. Note lessened surface immunoreaction of GHR by anti-GHRext-mAb in cells treated with GH at either 37 C (C) or 4 C (D). The data shown are representative of three such experiments.

unbound antibodies, cells were detergent solubilized, and protein G-Sepharose was added to the lysate to precipitate the antibodies. Eluted proteins were resolved by SDS-PAGE and immunoblotted with antiGHRcyt-AL47. This revealed that surface receptor im-

munoreactivity by anti-GHRext-mAb was dramatically reduced by prior treatment with GH (lane 2 vs. 1). As a control, aliquots of the cell extracts were resolved without immunoreaction and anti-GHRcyt-AL47 immunoblotted (lanes 3 and 4). This confirmed that GHR


was similarly abundant in the extracts, independent of prior GH treatment. Although the total GHR abundance was unchanged in Fig. 1C after 15 min GH treatment at 37 C, it is conceivable that ligand-induced internalization caused some clearing of surface GHR under those conditions and thereby could account for some of the loss of surface immunoreaction by anti-GHRext-mAb. To pursue this further, we sought conditions of stimulation that might allow GH engagement of the GHR but prevent significant internalization. We previously demonstrated (19) that GH treatment for a more prolonged period at 4 C (a temperature at which internalization is essentially blocked) allows GH binding and, although less than at 37 C, a degree of GH-induced GHR disulfide linkage. GHR disulfide linkage, although not necessary for receptor activation, reflects GH-induced receptor conformational changes associated with productive engagement (9, 10). In the experiment shown in Fig. 1D, serum-starved C14 cells were treated with (⫹) or without (⫺) GH for 90 min at 4 C. After this period, the cells were washed and reincubated with anti-GHRext-mAb (lanes 1 and 2) for 45 min at 4 C. After washing off unbound antibodies, cells were detergent solubilized and protein G-Sepharose was added to the lysate to precipitate the antibodies. Eluted proteins were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. As a control, aliquots of the cell extracts were resolved without immunoprecipitation and anti-GHRcyt-AL47 immunoblotted (lanes 3 and 4). This revealed that in cells treated with GH, anti-GHRext-mAb immunoreaction was again substantially reduced (lane 2 vs. 1), despite a lack of difference in total GHR abundance (lane 4 vs. 3). The same cell extracts were also resolved under nonreducing conditions (lanes 5 and 6), confirming that GH did indeed induce the appearance of the high-Mr disulfide-linked (dsl) form of the GHR under these conditions. Collectively, the findings in Fig. 1 indicate that anti-GHRext-mAb recognizes cell surface GHRs but loses its ability to immunoreact with the surface receptor when it is engaged by GH. Anti-GHRext-mAb was raised against a fusion protein that includes GST linked to the entire rbGHR ECD (diagrammed in Fig. 2A). This domain is comprised of two nearly equally sized regions referred to as subdomains 1 and 2, which are connected by a short hinge region. Because these subdomains form separate moieties within the receptor protein (4), we sought to determine whether anti-GHRext-mAb recognized either one preferentially. We constructed and bacterially expressed fusion proteins encoding receptor residues 1–128 (GST/GHR1–128) and 129–246 (GST/GHR129–246) for comparison with GST/GHR1–246, the immunogen for anti-GHRext-mAb. The fusions were designed to respect the boundaries of the subdomains, as defined from the hGHR ECD crystal structure (4), so as to maximize the likelihood that these proteins would maintain stability. Equal protein amounts of extract from bacteria expressing GST/GHR1–246, GST/GHR1–128, or GST/


GHR129–246 were resolved in duplicate by SDS-PAGE and immunoblotted with anti-GST (Fig. 2B, lanes 1–3) or anti-GHRext-mAb (lanes 4–6). Each fusion protein was recognized by anti-GST and migrated with its predicted Mr, suggesting substantial integrity of each protein in the bacterial lysates. However, only GST/GHR1–246 and GST/GHR129–246, but not GST/GHR1–128, were recognized by blotting with anti-GHRext-mAb (lanes 4 and 6 vs. 5). Similarly, immunoprecipitation of the fusions from the bacterial extracts with anti-GHRext-mAb followed by anti-GST immunoblotting revealed that GST/GHR1–128, despite being amply expressed, was not recognized by anti-GHRext-mAb, whereas the other two fusions were precipitated. These data indicate that the epitope(s) for anti-GHRext-mAb within the GHR ECD resides in subdomain 2 and not subdomain 1 or the hinge region. This is an important conclusion in that the majority of the residues involved in GH binding are found in subdomain 1, whereas the region of the GHR dimer interface in the GH-engaged GH-GHR2 complex is found in subdomain 2 (4). We attempted to further define one or more regions within subdomain 2 that might harbor an epitope for anti-GHRext-mAb by creating three fusion proteins to subdivide the subdomain. Subdomain 2 has seven ␤ sandwich strands, designated A, B, C, C⬘, E, F, and G, connected to each other by loops. In the crystal structure of the receptor, it is evident that these strands organize themselves such that together they form a sandwich with two antiparallel ␤ sheets, one with three strands (A, B, and E) and the other with four strands (C, C⬘, F, and G) (4). It is apparent that any division of the subdomain into fragments that by necessity include linear stretches of residues could not recreate these sheets (as strand E is between strands C⬘ and F in the linear sequence). Nonetheless, we constructed fusion proteins so as to respect the boundaries of strands and loops. The three GST fusions are diagrammed in Fig. 2C and included GHR residues 128–169 (the A strand, A-B loop, B strand, and B-C loop), 169–202 (the C strand, C-C⬘ loop, C⬘ strand, C⬘-E loop, and E strand), and 202–246 (the E-F loop, F strand, F-G loop, G strand, and juxtamembrane stem). Each was tested for its detectability by blotting and precipitation by anti-GHRext-mAb (Fig. 2, D and E) in the same fashion as were the subdomain 1- and subdomain 2-containing fusions in Fig. 2B. The three fusions were similarly well detected by anti-GST blotting at their expected Mr (Fig. 2D, lanes 2–4), indicating that each was stable in the bacterial expression system. Although equally well detected by anti-GST blotting as was GST/GHR129–246, none of the three fusions (GST/ GHR128–169, GST/GHR169–202, or GST/GHR202–246) were detectable compared with GST/GHR129–246 by anti-GHRext-mAb when all fusions were loaded in similar abundance on the gel (Fig. 2D), showing that reactivity was disrupted by further subdivision of the subdomain. This conclusion was further bolstered by the finding that two other fusions, GST/GHR128–202 and GST/GHR169–246, were also well detected by anti-



GST, but not detected by anti-GHRext-mAb (data not shown). Unlike the remainder of the ECD, little structural information is available about the GHR stem region (residues 238–246), which lies at the C terminus of subdomain 2 (4). This region links the ␤-sheets of the subdomain to the transmembrane portion of the receptor and harbors a site for regulated metalloprotease-mediated GHR cleavage that yields the shed receptor ECD (the GH binding protein; GHBP) (11–15). Because subdomain 2, which includes the stem region, reacts with anti-GHRext-mAb, we tested whether GHR mutants disrupted in the stem region could be recognized by the antibody by immunoprecipitation (Fig. 2E). We previously characterized several rabbit receptor mutants with internal deletions of three residues each within the stem region (15). These were referred to as ⌬237–239, ⌬240–242, and ⌬242–244 to indicate the deleted residues; in each case, GHR cell surface presentation, GH binding, and GH-induced signaling were intact for each mutant, but each was defective in undergoing phorbol ester-induced proteolysis (15). (Each of these mutants was in the backbone of the rbGHRdel 297–406 mutant, designated wild type (WT)⬘, which also has an internal deletion in the cytoplasmic domain that renders it defective in internalization.). Each mutant and the WT⬘ receptor were expressed in ␥2A-JAK2 cells. This cell line was derived from the same parental cell (␥2A) as was C14 (20); although it also stably expresses JAK2, ␥2A-JAK2, in contrast to C14, expresses no GHR and is thus a

Fig. 2. Anti-GHRext-mAb Recognizes GHR ECD Subdomain 2, But Not Subdomain 1 or the ECD Stem Region A and C, GST/GHR fusion proteins. The rbGHR ECD (residues 1–246) is diagrammed along with GST fusion proteins GST/GHR1–246, GST/GHR1–128, and GST/GHR129–246 (A). Subdomain 1, subdomain 2, the hinge region, and the stem region are indicated. Also indicated are residues involved in GH binding (open arrows) and the receptor dimerization interface (closed bars). Similar representation of GST129–246, GST128–169, GST169–202, and GST202–246 is presented in panel C, with indicated receptor dimerization interface residues and the stem region. B and D, Immunological detection of GST fusion proteins. Each of the fusion proteins diagrammed in

panels A and C were expressed in E. coli. Aliquots of the indicated extracted proteins were resolved by SDS-PAGE without immunoprecipitation (B, lanes 1–6; D, lanes 1–8) or immunoprecipitated with anti-GHRext-mAb and eluates were resolved by SDS-PAGE (B, lanes 7–9; D, lanes 9–12). Proteins were immunoblotted with anti-GST (B, lanes 1–3, 7–9; D, lanes 1–4 and 9–12) or anti-GHRext-mAb (B, lanes 4–6; D, lanes 5–8). Note that anti-GHRext-mAb specifically recognized GST/GHR1–246 and GST/GHR129–246 (but not GST/GHR1–128 or GST/GHR128–169, GST/GHR169–202, or GST/GHR202–246) by both immunoprecipitation and immunoblotting. The data shown are representative of three (B) and two (D) such experiments. E, Stem region mutants. ␥2A-JAK2 cells were transfected with rbGHR⌬297–406 (designated WT⬘), rbGHR⌬297–406 ⌬237–239 (⌬237–239), rbGHR⌬297–406 ⌬240–242 (⌬240–242), or rbGHR⌬297–406 ⌬242–244 (⌬242–244), as indicated, and serum-starved. Detergent extracts were immunoprecipitated with anti-GHRext-mAb (lanes 1–4) or beads only (lane 5) and eluates were resolved by SDS-PAGE. Aliquots of the same extracts were resolved without immunoprecipitation (lanes 6–10). GHRs were revealed by immunoblotting with anti-GHRcyt-AL47 (lanes 1–10). Note that each receptor stem region mutant was recognized by immunoprecipitation with anti-GHRext-mAb and that precipitation with this antibody was specific in this system [no GHR precipitated by beads only (lane 5)]. The positions of mature GHR (bracket) and precursor GHR (arrowhead) are indicated. The data shown are representative of two such experiments.


convenient cell in which to compare transiently transfected GHR mutants. Aliquots of detergent extract from each transfected population were immunoprecipitated with anti-GHRext-mAb or resolved without immunoprecipitation and immunoblotted with antiGHRcyt-AL47. Each receptor was found to be precipitated in proportion to its expression level (compare lanes 1–4 vs. 6–9). As a negative control, the WT⬘ receptor was not detected in immunoprecipitates in which no antibody was added (lane 5 vs. 10). Thus, none of the mutations in the stem region of the rbGHR ECD prevented recognition of the receptor by antiGHRext-mAb, leading us to conclude that an intact stem region does not contribute substantially to the epitope recognized by this antibody. Anti-GHRext-mAb Does Not Recognize a SignalingDefective GHR Dimerization Interface Mutant The so-called dimerization interface in the GHR ECD is an extensive region that includes six interspersed intermolecular bonds between the two receptors within the GH-bound complex. These are serine-145/aspartic acid-152, leucine-146/serine-201, threonine-147/ aspartic acid-152, histidine-150/asparagine-143, aspartic acid-152/tyrosine-200, serine-201/tyrosine-200 (4). Together, this interface occupies 500 Å2, as compared with roughly 1230 Å2 and 900 Å2 for the site 1-GHR and site 2-GHR interactions, respectively; thus, it is important for the overall stability of the assemblage. In a previous study, rbGHR mutants with changes in these residues were shown not to disrupt GH binding; however, some mutants were found to be unable to mediate GH-induced transcriptional signaling when stably expressed in Chinese hamster ovary cells (21). We examined the impact of the dimerization interface on the ability of anti-GHRext-mAb to detect the receptor by studying two GHR mutants, rbGHRH150D and rbGHR-T147K (21). rbGHR-H150D is a point mutation of histidine-150 to aspartate and rbGHR-T147K changes threonine-147 to lysine. Both residues 147 and 150 are known to be part of the dimerization interface, but mutation of residue 150 severely impairs GH-induced transcriptional signaling, whereas mutation of residue 147 is only marginally detrimental (21). We first tested the ability of these two mutant receptors to undergo acute GH-induced tyrosine phosphorylation when transiently expressed in COS-7 cells in comparison to the WT rbGHR (Fig. 3). COS-7 cells were cotransfected with plasmids encoding JAK2 and either the WT or mutant GHR and serumstarved cells were treated with (⫹) or without (⫺) GH for 15 min before detergent extraction. The extracts were immunoprecipitated with anti-GHRcyt-mAb and immunoblotted sequentially with anti-pTyr and antiGHRcyt-AL37, as indicated (Fig. 3A, upper and lower panels, respectively). GH treatment resulted in tyrosine phosphorylation of the receptor in cells in which WT rbGHR was expressed (Fig. 3A, upper panel, lane 2 vs.


1), consistent with our previous observations (9, 22– 24). Similarly, expression of rbGHR-T147K also allowed GH-induced GHR tyrosine phosphorylation (upper panel, lane 4 vs. 3), although to a lesser degree than that seen for WT rbGHR. In contrast, GH failed to induce receptor tyrosine phosphorylation in cells expressing rbGHR-H150D (upper panel, lane 6 vs. 5). Although rbGHR-T147K expression was somewhat less than that of WT rbGHR (possibly contributing to its somewhat diminished level of GH-induced tyrosine phosphorylation), it is clear that rbGHR-H150D tyrosine phosphorylation was undetectable despite its ample expression (lower panel, lanes 1–6). We also assessed GH-induced disulfide linkage of GHRs in cells expressing each of these GHR forms (Fig. 3B). GHR disulfide linkage, although not necessary for receptor activation, reflects GH-induced receptor conformational changes associated with productive engagement (9, 10). In this assay, cells stimulated with GH were lysed and extracts were resolved under nonreducing conditions (upper panel) before immunoblotting with anti-GHRcyt-AL47. GH induced the appearance of a high-Mr GHR form of the receptor in cells expressing either WT or T147K rbGHRs (lanes 2 vs. 1 and 4 vs. 3). However, the H150D rbGHR did not undergo disulfide linkage in response to GH treatment (lane 6 vs. 5). Similar GHR expression was verified by anti-GHRcyt-AL47 blotting of reduced extracts from the same samples (lower panel, lanes 1–6). These data suggested that, consistent with previous observations (21), mutation of residue 150 was more deleterious to GHR function than was mutation of residue 147. Furthermore, they validate the correlation between GH-induced GHR disulfide linkage and achievement of a receptor signaling conformation. The two dimerization interface mutants were next compared with WT for their ability to be immunoprecipitated by anti-GHRext-mAb (Fig. 3C). Serum-starved COS-7 cells expressing WT, rbGHR-T147K, or rbGHRH150D were harvested and detergent solubilized. Portions of the extracts were immunoprecipitated with either anti-GHRext-mAb or anti-GHRcyt-mAb and eluates of these precipitates were resolved by SDS-PAGE. Immunoblotting with anti-GHRcyt-AL47 revealed that both mutants were expressed and detected by antiGHRcyt-AL47 after anti-GHRcyt-mAb precipitation (Fig. 3C, lanes 4–6). However, only WT and rbGHR-T147K, but not rbGHR-H150D, were precipitated by anti-GHRext-mAb (Fig. 3C, lanes 1 and 2 vs. 3). Thus, the dimerization interface mutation that rendered rbGHRH150D unable to undergo conformational change and stimulate JAK2-mediated GHR tyrosine phosphorylation in response to GH also impaired the ability of this mutant to be recognized by anti-GHRext-mAb, even in the absence of GH stimulation. We note that, although detected by anti-GHRext-mAb, rbGHR-T147K was less well recognized than was WT. We do not yet completely understand this difference, but it may indicate that this mutation renders the dimerization



interface less intact than WT, but clearly not as disrupted as does mutation of residue 150. Collectively, these data, along with the mapping data in Fig. 2, indicate that the receptor dimerization interface forms the epitope for anti-GHRext-mAb or is required for another region of subdomain 2 to form the antibody’s epitope. Anti-GHRext-mAb Inhibits GH-Induced Signaling

Fig. 3. Analysis of Dimerization Interface Mutants A and B, Characterization of rbGHR-T147K and rbGHRH150D. A, GH-induced tyrosine phosphorylation. COS-7 cells were transfected with expression plasmids encoding JAK2 and either WT rbGHR (lanes 1 and 2), rbGHR-T147K (lanes 3 and 4), or rbGHR-H150D (lanes 5 and 6). Serumstarved cells were treated with vehicle (⫺) or GH (⫹) for 15 min before detergent extraction and immunoprecipitation with anti-GHRcyt-mAb. Eluates were resolved by SDS-PAGE and sequentially immunoblotted with anti-pTyr (upper panel) and anti-GHRcyt-AL37 (lower panel). The positions of the tyrosine phosphorylated GHR (pTyr GHR), the mature GHR (bracket), and the GHR precursor (arrowhead) are indicated. The data shown are representative of four such experiments. (B) GH-induced GHR disulfide linkage. Serum-starved COS-7 cells, transfected as in A, were treated with GH or vehicle for 15 min. Detergent extracts were resolved under either nonreduced (upper panel) or reduced (lower panel) conditions before immunoblotting with anti-GHRcyt-AL47. Note the GHinduced appearance of a high-Mr form of the GHR under nonreduced conditions for WT rbGHR and rbGHR-T147K, but not rbGHR-H150D. The positions of the dsl GHR, the mature GHR (bracket), and the GHR precursor (arrowhead) are indicated. The data shown are representative of three such experiments. C, Anti-GHRext-mAb fails to recognize rbGHR-H150D, but does recognize rbGHR-T147K. Serumstarved COS-7 cells expressing either WT rbGHR (lanes 1 and 4), rbGHR-T174K (lanes 2 and 5), or rbGHR-H150D (lanes 3 and 6) were detergent extracted and immunoprecipitated with either anti-GHRext-mAb (lanes 1–3) or antiGHRcyt-mAb (lanes 4–6). Eluates were resolved by SDSPAGE and immunoblotted with anti-GHRcyt-AL47. Note that

The data in Figs. 1–3 support the view that antiGHRext-mAb specifically recognizes the cell surface rbGHR in a conformationally sensitive manner that depends on the integrity of the dimerization interface within subdomain 2 of the ECD. Given the likely importance of GH-induced conformational changes within this interface for GH signal transduction, we investigated the effects of treatment of intact cells with anti-GHRext-mAb on GH-induced activation of intracellular signaling pathways (Fig. 4). Serum-starved C14 cells were pretreated with varying concentrations of anti-GHRext-mAb for 15 min at 37 C before treatment with GH for a further 15 min. Cells were then solubilized and proteins resolved by SDS-PAGE and immunoblotted with anti-pTyr-JAK2 (Fig. 5A, upper panel). This antibody recognizes phosphorylated tyrosine residues in the JAK2 kinase activation loop and the immunoblot signal thereby reflects the degree of JAK2 activation. As expected, GH treatment without prior exposure to anti-GHRext-mAb resulted in substantial JAK2 activation (lane 2 vs. 1). Pretreatment with antiGHRext-mAb, however, markedly inhibited GH-induced JAK2 activation (lanes 3–5 vs. 2). Notably, pretreatment with anti-GHRcyt-mAb, at a concentration comparable to the highest concentration of antiGHRext-mAb, failed to inhibit GH-induced JAK2 phosphorylation (lane 6 vs. 2). This is an important control for nonspecific effects of monoclonal antibody addition to the intact cells. The level of total JAK2 did not vary among the samples (not shown). Thus, the conformationally sensitive antibody could block the effect of GH on JAK2 activation. In other experiments (not shown), addition of anti-GHRext-mAb alone over a range of concentrations did not cause activation of JAK2 in this same assay. We also assessed similarly the effect of antiGHRext-mAb on STAT5 phosphorylation by immunoblotting with a phosphospecific anti-pTyrSTAT5 antibody (Fig. 5B). As for JAK2 activation, acute GHinduced STAT5 tyrosine phosphorylation was also inhibited by pretreatment with anti-GHRext-mAb, but not with anti-GHRcyt-mAb. As expected, STAT5 levels were not affected by antibody treatment (not shown).

rbGHR-H150D was not precipitated by anti-GHRext-mAb. The positions of mature GHR (bracket) and precursor GHR (arrowhead) are indicated. The data shown are representative of three such experiments.


Fig. 4. Inhibition of Acute GH Signaling by Anti-GHRext-mAb A–C, Serum-starved C14 cells were exposed for 15 min to the indicated concentrations of anti-GHRext-mAb (lanes 3–5), anti-GHRcyt-mAb (24 ␮g/ml; lane 6), or no antibody (lanes 1 and 2) before treatment with GH (500 ng/ml for 15 min; lanes 2–6) or vehicle (lane 1). Detergent extracts were resolved by SDS-PAGE under reduced (A and B) or nonreduced (C) conditions and immunoblotted with anti-pTyrJAK2 (A), antipTyrSTAT5 (B), or anti-GHRcyt-AL47 (C). The positions of pTyr JAK2, pTyr STAT5, the dsl GHR, and non-dsl GHR are indicated. Note inhibition of GH-induced JAK2 and STAT5 tyrosine phosphorylation and GH-induced GHR disulfide linkage by anti-GHRext-mAb pretreatment. The data shown are representative of five such experiments.

Notably, the anti-GHRext-mAb concentration dependences for inhibition of both JAK2 activation and STAT5 tyrosine phosphorylation were similar, suggesting the same mechanism of inhibition. To probe the mechanism further, we also examined whether the GH-induced formation of disulfide-linked GHRs was impacted by anti-GHRext-mAb pretreatment (Fig. 5C). Anti-GHRcyt-AL47 immunoblotting of nonreduced C14 cell detergent extracts revealed that GH-induced GHR disulfide linkage (lane 2 vs. 1) was indeed progressively inhibited by increasing concentrations of antiGHRext-mAb (lanes 3–5 vs. 2), whereas the total GHR abundance (dsl and non-dsl forms) was similar among the samples. Again, anti-GHRcyt-mAb had no effect on formation of the dsl GHR. This result supports the view that it is the binding of anti-GHRext-mAb to the GHR, rather than a spurious effect (on tyrosine kinase activation, for example), that accounts for the antibody’s inhibitory effect on GH signaling. In principle, however, the inhibition of GH-induced GHR disulfide linkage, JAK2 activation, and STAT5 tyrosine phosphorylation by anti-GHRext-mAb might be related to inhibition of GH binding to the GHR. Our previous results indicated that the GH-induced loss of anti-GHRext-mAb immunoprecipitation of the GHR was not mimicked by the GH antagonist, G120K, which binds the GHR, but fails to induce the receptor con-


Fig. 5. Effect of Anti-GHRext-mAb on Surface 125I-GH Binding Serum-starved C14 cells were pretreated with antiGHRext-mAb at the indicated concentrations for 30 min at 37 C and then incubated with 125I-hGH (50,000 cpm; ⬃25 pM) at room temperature for 1 h. Within each experiment, the specific 125I-hGH binding was determined in triplicate as described in Materials and Methods. The data from n ⫽ 3 (1.5 ␮g/ml), n ⫽ 2 (3 ␮g/ml), n ⫽ 2 (9 ␮g/ml), and n ⫽ 3 (18 ␮g/ml) experiments are plotted (mean ⫾ SE), in each case relative to the binding detected in the absence of antibody pretreatment (considered 100%) within the same experiment. Note that maximum binding inhibition by antibody pretreatment was roughly 50%.

formational changes required for signaling (9). This led us to conclude that anti-GHRext-mAb did not substantially compete for GH binding. However, to address this further in the context of our current findings, we performed 125I-hGH binding experiments with C14 cells (Fig. 5). Serum-starved cells in monolayer were preincubated for 30 min at room temperature with varying concentrations of anti-GHRext-mAb or, as a control, a high concentration of anti-GHRcyt-mAb before continued incubation for 1 h with 125I-hGH (50,000 cpm per sample; 25 pM) in the presence or absence of excess unlabeled hGH to determine nonspecific binding. Specific 125I-hGH cell surface binding was determined after washing and solubilization of the cells by ␥-counting and subtraction of nonspecific binding. Radiolabeled GH binding was somewhat inhibited by increasing concentrations of anti-GHRext-mAb. However, it is notable that the inhibition was incomplete even at the highest concentrations of antibody used (concentrations far above those that were completely inhibitory in the experiments in Fig. 4). Furthermore, it is also important that the concentration of radiolabeled GH present in the binding experiments (⬃0.55 ng/ml; 25 pM) was far less than that to which cells were exposed in the signaling experiments (500 ng/ml; 22.7 nM). Thus, the molar ratio of anti-GHRext-mAb:GH was much greater in the binding experiments than in the signaling experiments; yet, the inhibition of binding of anti-GHRext-mAb was far less efficient than its inhibition of signaling. These data suggest that the effects of the antibody on GH signaling are not likely explained solely by its ability to inhibit GH binding, but instead reflect its blockade of GH-induced conformational changes in the receptor required for activation.


Effects of Anti-GHRext-mAb on Inducible GHR Proteolysis We previously demonstrated that inducible GHR proteolysis is catalyzed by a metalloprotease activity and may be a regulator of cellular GH sensitivity (11–13, 15, 25). Furthermore, this activity is likely responsible for generation of GH binding protein in humans and other species (26). The data in Figs. 4 and 5 demonstrated that GH-induced receptor activation was prevented by anti-GHRext-mAb by a mechanism other than inhibition of GH binding. Despite our findings that residues in the stem region of the ECD that includes the receptor cleavage site do not contribute significantly to the epitope(s) recognized by anti-GHRext-mAb (Fig. 2E), we similarly examined whether pretreatment with antiGHRext-mAb would affect the ability of the GHR to be inducibly proteolyzed (Fig. 6). Serum-starved C14 cells were pretreated with varying concentrations of antiGHRext-mAb for 30 min before treatment with phorbol12-myristate-13-acetate (PMA) or its vehicle for 30 min. Cells were harvested, and detergent soluble proteins were resolved and immunoblotted with antiGHRcyt-AL47. As we have previously shown for several cell types (12–15, 17), PMA treatment caused a dra-

Fig. 6. Effect of Anti-GHRext-mAb on Inducible GHR Proteolysis A, Serum-starved C14 cells were pretreated for 30 min with the indicated concentrations of anti-GHRext-mAb (lanes 3–8), anti-GHRcyt-mAb (24 ␮g/ml; lanes 9 and 10), or no antibody (lanes 1 and 2), followed by treatment for 30 min with vehicle (⫺) or PMA (⫹). Detergent extracts were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of mature GHR (bracket), GHR precursor (arrowhead), and GHR remnant (arrow) are indicated. B, Immunoblots such as that shown in A were evaluated densitometrically to determine the PMA-induced loss of mature GHR. The data from n ⫽ 3 (0 ␮g/ml), n ⫽ 3 (1.5 ␮g/ml), n ⫽ 2 (3 ␮g/ml), n ⫽ 3 (6 ␮g/ml), and n ⫽ 3 (12 ␮g/ml) experiments are plotted (mean ⫾ SE), in each case indicating the relative loss of GHR in the presence vs. absence of PMA.


matic loss of the full-length GHR (indicated by a bracket) and the appearance of a roughly 65-kDa protein (indicated by an arrow) reactive with the anti-GHR serum directed at the cytoplasmic domain (Fig. 7A, lane 2 vs. 1). We previously termed the latter protein the GHR remnant, which contains the receptor cyto-

Fig. 7. Inducible Proteolysis of Dimerization Interface Mutants A, Activation of dimerization interface mutants in ␥2AJAK2 cells. Serum-starved ␥2A-JAK2 cells transfected with either WT rbGHR (lanes 1,2), rbGHR-T147K (lanes 3 and 4), or rbGHR-H150D (lanes 5 and 6) were treated with (⫹) or without (⫺) GH for 15 min before detergent extraction and sequential immunoblotting with anti-pTyrJAK2 (upper panel) and anti-GHRcyt-AL47 (lower panel). The positions of pTyrJAK2 (arrow), mature GHR (bracket), and precursor GHR (arrowhead) are indicated. The data shown are representative of two such experiments. B, Inducible proteolysis of dimerization interface mutants in ␥2A-JAK2 cells. Serum-starved ␥2A-JAK2 cells transfected with either WT rbGHR (lanes 1 and 2), rbGHR-T147K (lanes 3 and 4), or rbGHR-H150D (lanes 5 and 6) were treated with (⫹) or without (⫺) PMA for 30 min before detergent extraction and immunoblotting with anti-GHRcyt-AL47. The positions of mature GHR (bracket), GHR precursor (arrowhead), and GHR remnant (arrow) are indicated. The data shown are representative of three such experiments. C, Effects of GH and anti-GHRext-mAb on inducible proteolysis of WT rbGHR and rbGHR-H150D. Serumstarved ␥2A-JAK2 cells transfected with either WT rbGHR (lanes 1–6) or rbGHR-H150D (lanes 7–12) were pretreated with either vehicle (lanes 1, 2, 7, and 8), anti-GHRext-mAb (lanes 3, 4, 9, and 10), or GH (500 ng/ml; lanes 5, 6, 11, and 12) before treatment for 30 min with PMA (lanes 2, 4, 6, 8, 10, and 12) or vehicle (lanes 1, 3, 5, 7, 9, and 11). Detergent extracts were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The positions of mature GHR (bracket) and GHR precursor (arrowhead) are indicated. The data shown are representative of three such experiments.


plasmic and transmembrane domains as well as the eight ECD residues that remain after proteolysis and shedding of the majority of the ECD as the GHBP (11, 15). As the concentration of anti-GHRext-mAb added to the cells during the pretreatment period was increased, there was no appreciable effect on the basal abundance of either receptor or remnant (lanes 3, 5, and 7 vs. 1), but there was a notable progressive inhibition of PMA-induced receptor loss and remnant accumulation (lanes 3 and 4, 5 and 6, and 7 and 8 vs. 1 and 2). PMA-induced receptor cleavage was nearly completely inhibited by the presence of anti-GHRextmAb at 12 ␮g/ml, but anti-GHRcyt-mAb failed to inhibit proteolysis (lanes 9 and 10 vs. 7 and 8). Several such experiments were analyzed denstiometrically and the pooled results are presented in Fig. 6B. These data suggest that anti-GHRext-mAb specifically inhibits inducible metalloproteolysis of the rbGHR, even though it does not directly bind the receptor at the cleavage site. We anticipated that the ability of anti-GHRext-mAb to inhibit inducible receptor proteolysis would be lost in the rbGHR dimerization mutant, H150D. To test this, we examined proteolysis of rbGHR-T147K, rbGHRH150D, and WT, using the ␥2A-JAK2 cell line. We first verified that each exhibited the same signaling properties in these cells as we observed in COS-7 cells in Fig. 3. ␥2A-JAK2 cells were transiently transfected with expression vectors encoding either WTrbGHR, rbGHR-T147K, or rbGHR-H150D, serum starved, and treated with GH or vehicle for 15 min (Fig. 7A). Detergent extracts were resolved and immunoblotted with anti-pTyrJAK2, revealing that GH induced JAK2 activation in cells expressing WT and rbGHR-T147K, but not rbGHR-H150D (upper panel, lanes 2 and 4 vs. 6). Transfected GHR abundance (Fig. 7A, lower panel) and endogenous JAK2 levels (data not shown) were determined to be similar in each condition by immunoblotting. These results paralleled those in COS-7 cells and indicated that rbGHR-H150D is also deficient in GH-induced signaling in the context of ␥2A-JAK2 cells. The susceptibility of each receptor in this system to inducible proteolysis was assessed by treatment of transiently transfected cells with PMA or vehicle followed by anti-GHRcyt-AL47 immunoblotting of detergent cell extracts (Fig. 7B). This revealed that both mutants, like WT rbGHR, were inducibly proteolyzed in response to PMA, indicating that mutations in the dimerization interface do not interfere with the proteolytic process, independent of whether they interfere with GH-induced signaling. WTrbGHR and rbGHR-H150D (the signaling-incompetent dimerization interface mutant) were further compared with regard to inducible proteolysis in the experiment shown in Fig. 7C. As expected, PMA treatment of cells transiently expressing WTrbGHR caused receptor loss (lane 2 vs. 1), and this effect was blocked by preincubation with anti-GHRext-mAb (lanes 3 and 4 vs. 1 and 2). We previously demonstrated that exposure of cells to GH inhibited subsequent inducible


receptor proteolysis, an effect believed to indicate that GH-induced conformational changes rendered the receptor less susceptible to metalloprotease-mediated cleavage (14). This inhibitory effect of GH was again seen when WTrbGHR was transiently expressed in ␥2A-JAK2 cells (lanes 5 and 6 vs. 1 and 2). rbGHRH150D, despite being incompetent for GH-induced signaling, was inducibly proteolyzed in response to PMA in a fashion indistinguishable from WTrbGHR (lanes 7 and 8 vs. 1 and 2). However, unlike WTrbGHR, neither anti-GHRext-mAb nor GH pretreatment blocked PMA-induced rbGHR-H150D proteolysis (lanes 9 and 10 and 11 and 12 vs. 3 and 4 and 5 and 6). The lack of GH protection of rbGHR-H150D from proteolysis is consistent with the notion that a GH-induced receptor conformational change is required if protection is to be afforded. The inability of anti-GHRext-mAb to prevent rbGHR-H150D proteolysis is in accordance with its inability to recognize the same mutant receptor by immunoprecipitation (Fig. 3C); furthermore, it bolsters the conclusion that prevention of WTrbGHR cleavage by anti-GHRext-mAb is not due to direct interference with the stem region cleavage site. A Fab Fragment Mimics the Effects of Intact anti-GHRext-mAb Our data indicate that anti-GHRext-mAb specifically inhibits GH-induced signaling, although much less effectively reducing cell surface GH binding. Furthermore, this antibody also specifically inhibits PMAinduced GHR proteolysis. Because anti-GHRext-mAb appears to recognize the GHR in a conformationally sensitive fashion, we considered whether some of its effects might be exerted by the non-antigen-recognizing regions of the antibody. For example, could the inhibition of anti-GHRext-mAb of receptor proteolysis be due its binding to its epitope (e.g. the dimerization interface) and sterically hindering metalloprotease access to the stem region cleavage site, simply by virtue of the presence of the antibody rather than induction of a receptor conformational change? To address this issue, we subjected anti-GHRext-mAb to papain cleavage and recovered the resulting Fab fragments. AntiGHRext-mAbFab was tested for its effects on GH signaling, GH binding, and inducible GHR proteolysis. Pretreatment of C14 cells with anti-GHRext-mAbFab before GH treatment markedly reduced GH-induced JAK2 tyrosine phosphorylation (Fig. 8A, upper panel), STAT5 tyrosine phosphorylation (Fig. 8B), and GHR disulfide linkage (Fig. 8A, lower panel). Anti-GHRext-mAbFab pretreatment also only partially inhibited 125I-GH surface binding to C14 cells (Fig. 8C) and markedly inhibited PMA-induced GHR proteolysis (Fig. 8D). It is useful to compare the relative effectiveness of anti-GHRext-mAbFab vs. anti-GHRext-mAb in these experiments. The expected molecular mass of a Fab fragment would be roughly one third that of an intact antibody molecule; thus, it would be anticipated that an equimolar effect of anti-GHRext-mAbFab might be



achieved with roughly one third the concentration (in micrograms per milliliter) as that of anti-GHRext-mAb. Notably, the inhibitory effect of anti-GHRext-mAbFab on GH-induced STAT5 tyrosine phosphorylation at 2 ␮g/ml was similar to that of anti-GHRext-mAb at 6 ␮g/ml (Fig. 8B, lanes 3 and 4 vs. 2). Likewise, the antiGHRext-mAbFab concentration dependencies for inhibition of 125I-GH binding and PMA-induced GHR loss were very similar to those for anti-GHRext-mAb for these effects, but shifted by roughly a factor of three (compare Figs. 8C vs. 5 and 8D vs. 6B). These data suggest that the inhibitory effects of anti-GHRext-mAb are not related to the Fc region of the molecule.

DISCUSSION

Fig. 8. A Fab Fragment Mimics the Effects of AntiGHRext-mAb on GH Signaling, GH-Induced GHR Disulfide Linkage, Surface 125I-GH Binding, and Inducible GHR Proteolysis A and B, GH signaling and GH-induced GHR disulfide linkage. Serum-starved C14 cells were exposed for 15 min to the indicated concentrations of anti-GHRext-mAbFab (A, lanes 3 and 4; B, lane 3) or anti-GHRext-mAb (B, lane 4) or no antibody (A and B, lanes 1 and 2) before treatment with GH (500 ng/ml for 15 min; A and B, lanes 2–4) or vehicle (A and B, lane 1). Detergent extracts were resolved by SDS-PAGE under reduced (A, upper panel, and B) or nonreduced (A, lower panel) conditions and immunoblotted with anti-pTyrJAK2 (A, upper panel), anti-GHRcyt-AL47 (A, lower panel), or anti-pTyrSTAT5 (B). The positions of pTyr JAK2, pTyr STAT5, the dsl GHR, and non-dsl GHR are indicated. Note inhibition of GH-induced JAK2 and STAT5 tyrosine phosphorylation and GH-induced GHR disulfide linkage by antiGHRext-mAbFab pretreatment. The data shown are representative of two such experiments each. C, Surface 125I-GH binding. Serum-starved C14 cells were pretreated with antiGHRext-mAbFab at the indicated concentrations for 30 min at 37 C and then incubated with 125I-hGH (50,000 cpm; ⬃25 pM) at room temperature for 1 h. Specific 125I-hGH binding was determined in triplicate and displayed as mean ⫾ SE relative to the binding detected in the absence of Fab fragment pretreatment (considered 100%). The experiment shown is representative of four independent experiments. D, Inducible GHR proteolysis. Serum-starved C14 cells were pretreated for 30 min with the indicated concentrations of antiGHRext-mAbFab, followed by treatment for 30 min with vehicle or PMA. Detergent extracts were resolved by SDS-PAGE and

Despite intimate knowledge of the structure of the GHR ECD, our understanding of the mechanisms of GH-induced receptor triggering is incomplete. Current views favor the likelihood that unliganded GHRs exist, at least to some degree, as homodimers and that GH binding causes (as yet unspecified) structural changes in the preformed dimers to allow an active signaling conformation to be achieved (7, 8, 27, 28). Because the crystal structure of the GH-engaged GHR dimer specifies close approximation of the dimerization interfaces of subdomain 2 of the ECD (4), it may follow that the GH-induced changes at least partly involve fostering this association and the consequent productive interaction of receptor cytoplasmic domains and their associated JAK2 tyrosine kinase molecules. The evidence for conformational changes being critical for GH-induced GHR activation has emerged in part from studies of anti-GHR ECD antibodies that are stimulatory. Mellado et al. (27) described an IgM monoclonal antibody reactive with the inter-subdomain hinge region. Independent of GH stimulation, this antibody promoted GHR-mediated activation of intracellular tyrosine phosphorylation and cellular proliferation and treatment with GH enhanced the antibody’s recognition of GHR, suggesting that the antibody mimicked a GH-induced change in receptor conformation. Similarly, a separate stimulatory anti-GHR ECD monoclonal antibody was shown to lose its stimulatory effect upon mutation of a loop in subdomain 2 that has been implicated as undergoing GH-induced conformational change (8, 29). Whether these supposed GH-induced changes in GHR structure are like those believed to occur for the erythropoietin (Epo) receptor, in response to Epo (30, 31) is as yet unknown. However, key differences, such as the rela-

immunoblotted with anti-GHRcyt-AL47. Immunoblots were evaluated densitometrically to determine the PMA-induced loss of mature GHR. The data from n ⫽ 3 experiments are plotted (mean ⫾ SE), in each case indicating the relative loss of GHR in the presence vs. absence of PMA.


tively much smaller dimerization interface in the Epo receptor, make comparisons difficult to draw (28). In this study, we further characterized a separate monoclonal anti-GHR ECD antibody, anti-GHRext-mAb, that is not stimulatory of GHR signaling. Rather, we defined it as conformationally sensitive in its recognition of the GHR and inhibitory of several GHRmediated phenomena. Our initial characterization of anti-GHRext-mAb indicated that its ability to immunoprecipitate the GHR was markedly lessened by pretreatment of cells with GH (14), a phenomenon confirmed in a different cell type and extended to consideration of the cell surface receptor in the current study in Fig. 1. G120K, a GH antagonist that binds GHR but fails to activate signaling, failed to itself inhibit anti-GHRext-mAb immunoprecipitation and antagonized the inhibitory effect of GH on anti-GHRext-mAb immunoprecipitation (14). It was thus seen as unlikely that the inhibitory effect of GH was due to its direct blockade of anti-GHRext-mAb recognition by virtue of the GH binding site being also the anti-GHRext-mAb epitope (14). Our current experiments in Fig. 2, B and D, support this view in that anti-GHRext-mAb immunoreactivity is present in subdomain 2 (which includes the dimerization interface), rather than subdomain 1 (which harbors most of the residues involved in GH binding). In these experiments, we note that the same results were obtained when anti-GHRext-mAb was used to detect the fusion proteins by both immunoprecipitation and immunoblotting. Both domains (1 and 2) are each comprised of several ␤ sandwich strands, which collectively organize into a sandwich of two ␤ sheets (4). The finding that separate pairs of these strands were not recognized by anti-GHRext-mAb in immunoblots and immunoprecipitation favors the view that its epitope is complex, requiring precise organization of these ␤ strands. This is similar to the situation with MAb 263, a monoclonal antibody that recognizes a complex GHR extracellular subdomain 1 epitope comprised of 20 amino acids distributed in a discontinuous manner (32). In the current study (Fig. 3, A–C), we showed that a receptor mutated within the dimerization interface that is functionally impaired in undergoing GH-induced signaling (rbGHR-H150D) is not recognized by antiGHRext-mAb, even in the absence of GH stimulation. Yet, another dimerization interface mutant (rbGHRT147K) retains the capacity (albeit to a reduced extent) to be detected by anti-GHRext-mAb. We find this result interesting in that, unlike rbGHR-H150D, mutation of residue 147 [even though it is also a point of dimer contact in the interface (4)] did not render the receptor incapable of activation or of undergoing GH-induced disulfide linkage (Ref. 21 and Fig. 3, A and B). Thus, only disruption of the dimerization interface residue that led to complete inability of the receptor to conformationally change and become activated was associated with complete loss of anti-GHRext-mAb reactivity. Considering these findings in the context of


the fusion protein-mapping experiments discussed above, it is plausible that the actual epitope for antiGHRext-mAb includes H150, but not T147, within the dimerization domain or that interface domain intactness is required for formation of the epitope at another region within the receptor ECD subdomain 2. Further studies will be necessary to discriminate between the latter two possibilities. However, the results in Fig. 2E indicate that residues in the ECD stem region [which, although nearby to the dimerization interface in the GHR crystal structure, are spatially distinct from it (4)] are not components of the anti-GHRext-mAb epitope. Our data clearly indicate that anti-GHRext-mAb can recognize the rbGHR on the surface of cells (Fig. 1) and, when applied to cells, the antibody or its Fab fragment specifically blocks proximal aspects of GH signaling (JAK2 and STAT5 tyrosine phosphorylation) (Figs. 4, A and B, and 8A). Furthermore, this blockade was accompanied by inhibition of GH-induced GHR disulfide linkage (Fig. 4C for the antibody; Fig. 8B for the Fab fragment). Receptor disulfide linkage via the free sulfhydryl group of cysteine-241 (the only unpaired ECD cysteine in the GHR) in the ECD stem region is a GH-induced event that corresponds directly to the induction of GH signaling in a variety of cell types, very likely by reflecting the GH-induced conformational changes in constitutive receptor dimers that convert them into dimers competent for signal transduction (7, 9, 10). Furthermore, our 125IhGH cell surface binding assays (Figs. 5 and 8C) showed that anti-GHRext-mAb or Fab fragment pretreatment only partially inhibited GH binding and that the molar ratio of antibody or Fab to GH required for even partial binding inhibition was far greater than that leading to complete inhibition of GH-induced GHR triggering and signaling. Thus, we favor the interpretation that anti-GHRext-mAb exerts its inhibitory effect on GH signaling by not allowing GH-induced receptor conformational changes to occur, rather than by either directly blocking GH binding or impairing selectively the ability of conformationally competent GHRs to activate JAK2. It is, of course, possible that antiGHRext-mAb, by inhibiting apposition of dimerization domain interfaces, prevents stabilization of the ternary complex (GH:GHR2) (6), and decreases equilibrium binding. However, if this were so, one would expect a doubling in sites available for 125I-GH binding in 1:1 complexes, as observed for MAb5, which recognizes the dimerization domain (33, 34). Our characterization of anti-GHRext-mAb may also help us to better understand the effects of GH-induced conformational change on the sensitivity of the GHR to inducible metalloproteolysis. Stimuli such as phorbol ester, platelet-derived growth factor, and serum can in a variety of cell systems cause metalloprotease-mediated GHR cleavage in the perimembranous ECD stem region (11–15, 17, 35). This can result in the shedding of the ECD as the GHBP and modulates cellular GH sensitivity (12, 13, 15). We previously demonstrated that pretreatment with GH lessens the subsequent


proteolysis of the receptor induced by PMA stimulation (14). The inhibitory effect of GH on GHR proteolysis is not due to GH signaling because it occurs even in the absence of JAK2; nor is it related to GH-induced GHR down-regulation because a receptor mutant lacking the cytoplasmic domain region required for internalization is still desensitized to inducible proteolysis by GH (14). Rather, as supported by the finding that the G120K GH antagonist did not inhibit proteolysis but did compete with GH for this effect, the inhibition appeared more related to GH induction of conformational changes in the GHR dimer (14). Indeed, the data in Fig. 7 of this report support this conclusion in that rbGHR-H150D, which apparently cannot undergo such conformational change, was inducibly cleaved in response to PMA and, unlike WT rbGHR, GH did not prevent this rbGHR-H150D cleavage. In this context, we find it interesting that antiGHRext-mAb or Fab fragment pretreatment specifically inhibited PMA-induced WT rbGHR proteolysis (Figs. 6, A and B, 7C, and 8C). Although it did not function as a GH agonist in terms of triggering signaling, antiGHRext-mAb mimicked GH with regard to inhibiting receptor proteolysis. However, it is unclear to what degree, if any, that the mechanisms of GH and the effects of anti-GHRext-mAb on GHR proteolysis are similar. Anti-GHRext-mAb does not appear to interact with the stem region that harbors the actual cleavage site and its inhibitory potential is lost on rbGHR-H150D, to which it does not bind. One possible mechanism of inhibition might be that anti-GHRext-mAb binding to the dimerization interface (or a region influenced by the dimerization interface) alters the conformation of the GHR dimer in such a way as to make stem region cleavage less likely. In this respect, GH may also change the dimerization interface to similarly lessen the susceptibility of the stem region cleavage site, but in so doing may in addition induce changes required for GHR triggering that are not allowed by anti-GHRext-mAb binding. Another unrelated potential mechanism of anti-GHRext-mAb inhibition of receptor proteolysis could be that antibody binding to its epitope (e.g. the dimerization interface) may sterically hinder metalloprotease access to the stem region cleavage site, simply by virtue of the presence of the antibody rather than induction of a receptor conformational change. Discrimination among these and other possible mechanisms of anti-GHRext-mAb inhibition of GHR proteolysis and assessment of any mechanistic similarities with GH in this respect will await further studies. However, our finding that the Fab fragment inhibits GHR proteolysis with very similar (molar) concentration dependence as anti-GHRext-mAb argues that any such steric hindrance would not be accounted for by the antibody’s Fc region. GH has important somatogenic and metabolic effects at the liver and other target tissues (1). With chronically increased levels, GH causes the characteristic stigmata and organ damage of acromegaly


(36). Furthermore, even at normal levels, GH may participate in mediation of pathologic changes, such as ischemia-induced neovascularization of the retina (37) and diabetes-related nephropathy (38). GH antagonists have proven useful clinically and experimentally in ameliorating these deleterious effects of GH (37–40). The studies described herein suggest that anti-GHRext-mAb and its Fab fragment can inhibit GH-induced signaling and GHR proteolysis in cell culture model systems. Further studies delineating more precisely the mechanisms of these effects may suggest whether this antibody or Fab may also have utility in vivo to modulate effects of GH and serve as a potential therapeutic agent. In addition, our current studies emphasize that, in addition to its ability to prevent the receptor from achieving the activated state, anti-GHRext-mAb loses recognition of the GHR when in its activated conformation and can subtly discriminate between receptors mutated within the dimerization interface on the basis of their capability for activation. These properties will make it an attractive tool in future studies of the mechanisms of GHinduced GHR activation, an area of major current interest in GH and cytokine research.

MATERIALS AND METHODS Materials PMA and routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Recombinant hGH was kindly provided by Eli Lilly Co. (Indianapolis, IN). Zeocin was purchased from Invitrogen Life Technologies (Carlsbad, CA). 125I-hGH (specific activity 85–130 ␮Ci/␮g) was purchased from PerkinElmer (Norwalk, CT). Cells, Cell Culture, and Transfection COS-7 cells were grown in DMEM (1 g/liter glucose) (Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum (Biofluids, Camarillo, CA), 50 ␮g/ml gentamicin sulfate, 100 U/ml penicillin, and 100 ␮g/ml streptomycin. C14 cells (previously referred to as ␥2A-GHR/JAK2) have been described (16). In brief, C14 is a stable cell line (clone 14) resulting from expression of the rabbit GHR and murine JAK2 in the JAK2-deficient ␥2A human fibrosarcoma cell line (41). C14 cells were maintained in DMEM (1 g/liter glucose) supplemented with 10% fetal bovine serum, 50 ␮g/ml gentamicin sulfate, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 200 ␮g/ml G418, 100 ␮g/ml hygromycin B, and 100 ␮g/ml Zeocin. ␥2A-JAK2 cells (20) were maintained in the same medium as C14 cells, but without hygromycin B. Transient transfection of COS-7 cells was performed at 75% confluency using the calcium phosphate precipitation method, as described previously (42, 43) in 100- ⫻ 20-mm dishes (Becton Dickinson Labware, Franklin Lakes, NJ). Each dish was transfected with 15 ␮g of GHR-expressing plasmids along with 10 ␮g pSX JAK2. Transient transfection of ␥2A-JAK2 cells was accomplished with LipofectAMINE Plus (Invitrogen Life Technologies) according to the manufacturer’s protocol, as previously described (16).


Plasmid Construction and Preparation of GST Fusion Proteins The plasmid encoding GST/GHR1–246 [GST N-terminal to residues 1–246 of the rabbit GHR (the entire ECD)] has been previously described (9, 12, 42). Analogous plasmids encoding rabbit GHR residues 1–128, 129–246, 128–169, 169–202, 202–246, 128–202, and 169–246 were generated by PCR subcloning into the EcoRI and XhoI sites of the pGEX-4T-1 vector (PCR primer sequences are available upon request). GST fusion proteins were expressed in Escherichia coli and purified as described previously (22). Expression plasmids for WT rbGHR, rbGHR-T147K, and rbGHR-H150D in the pECE vector have been described previously (21), as were pSXJAK2 (24) and pcDNA expression plasmids encoding WT⬘ (rbGHRdel 297–406), ⌬237–239, ⌬240–242, and ⌬242–244 (15). Antibodies The anti-p-JAK2 state-specific antibody reactive with JAK2 that is phosphorylated at residues Y1007 and Y1008 (reflective of JAK2 activation), anti-pTyr monoclonal antibody (4G10), and monoclonal anti-GST were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Rabbit anti-pTyrSTAT5 polyclonal antibody (raised against a phosphopeptide surrounding phosphorylated Tyr694 of murine STAT5 in both STAT5A and STAT5B) was obtained from Zymed Laboratories, Inc. (San Francisco, CA). The rabbit polyclonal antiserum, anti-GHRcyt-AL47, raised against a bacterially expressed N-terminally His-tagged fusion protein incorporating hGHR residues 271–620 [the entire cytoplasmic domain (2)], has been previously described (14). Anti-GHRcyt-AL37 was raised against a bacterially expressed GST fusion with hGHR residues 271–620, as described (44). Anti-GHRcyt-mAb is a mouse monoclonal antibody (IgG2b␬) directed against a bacterially expressed GST fusion protein incorporating hGHR residues 271–620 and has been previously described (9). Anti-GHRext-mAb, a mouse monoclonal antibody (IgG1␬) directed against a bacterially expressed GST fusion protein incorporating rabbit GHR residues 1–246, has been previously described (9, 12, 42). Both monoclonal anti-GHR antibodies were purified from hybridoma supernatant using protein G-Sepharose (at the UAB Multipurpose Arthritis Center Hybridoma Core facility). Preparation of anti-GHRext-mAbFab was carried out at Rockland Immunochemicals, Inc. (Gilbertsville, PA). In brief, 110 mg of purified antibody was subjected to papain digestion, followed by selective precipitation to separate Fab from Fc components and undigested antibody. After extensive dialysis, assay by immunoelectrophoresis resulted in a single precipitin arc against antimouse IgG F(ab⬘)2. No reaction was observed against antimouse IgG Fc. The yield of purified Fab fragment was roughly 13 mg at a stock concentration of 0.82 mg/ml in 0.02 M potassium phosphate/0.15 M NaCl (pH 7.2). Cell Stimulation, Protein Extraction, Immunoprecipitation, Electrophoresis, and Immunoblotting Serum starvation was accomplished by substitution of 0.5% (wt/vol) BSA (fraction V, Roche Molecular Biochemicals, Indianapolis, IN) for serum in culture media for 16–20 h before experiments. Unless otherwise noted, stimulations were performed at 37 C. Details of the hGH (500 ng/ml) and PMA (at 1 ␮g/ml) treatment protocols have been described (14–16). Briefly, adherent cells were stimulated in binding buffer [BB, consisting of 25 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 0.1% (wt/vol) BSA, and 1 mM dextrose] or DMEM (1 g/liter glucose) with 0.5% (wt/vol) BSA. Stimulations were terminated by washing the cells once with ice-cold PBS in the presence of 0.4 mM sodium orthovanadate (PBSvanadate) and then harvesting by scraping in cold PBS-


vanadate. Pelleted cells were collected by brief centrifugation. For each cell type, pelleted cells were solubilized for 15 min at 4 C in lysis buffer [1% (vol/vol) Triton X-100, 150 mM NaCl, 10% (vol/vol) glycerol, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM benzamidine, and 10 ␮g/ml aprotinin], as indicated. After centrifugation at 15,000 ⫻ g for 15 min at 4 C, the detergent extracts were electrophoresed under reducing or nonreducing conditions or subjected to immunoprecipitations, as indicated. For experiments in which the monoclonal antibodies and Fab fragment were tested for effects on cell signaling and GH binding, the indicated concentrations of purified antibodies or Fab fragment were added directly to serum-starved cells at 37 C for the pretreatment durations indicated in the figure legends. For standard immunoprecipitation of the GHR with the monoclonal anti-GHRcyt-mAb or anti-GHRext-mAb antibodies, 5 ␮g or 3 ␮g, respectively, of purified antibody per precipitation was added to detergent cell extracts. Protein-G Sepharose (Amersham Biosciences, Piscataway, NJ) was used to adsorb immune complexes. For surface immunoreaction, serum-starved cells were treated for 15 min at 37 C or 90 min at 4 C, as indicated, with or without GH (500 ng/ml), after which antibody was added directly to cells at 4 C for 45 or 60 min, as indicated. Thereafter, cells were washed three times with cold PBS and detergent-solubilized and antibodies were adsorbed on Protein-G Sepharose. In all cases, after extensive washing, sodium dodecyl sulfate sample buffer eluates were resolved by SDS-PAGE and immunoblotted, as indicated. Resolution of proteins by SDS-PAGE, Western transfer of proteins, and blocking of Hybond-ECL (Amersham, Inc.) with 2% BSA were performed as previously described (9, 15, 16). Immunoblotting with anti-GHRcyt-AL47 (1:2000–1: 4000), anti-GHRcyt-AL37 (1:2000), anti-pTyrJAK2 (1:1000), anti-pTyrSTAT5 (1:1000), anti-GST (1:1000), or anti-pTyr (1: 4000) with horseradish peroxidase-conjugated antimouse or antirabbit secondary antibodies (1:10000–1:15000) and detection reagents (SuperSignal West Pico Chemiluminescent Substrate) (all from Pierce, Rockford, IL) and stripping and reprobing of blots were accomplished according to manufacturers’ suggestions. 125

I-hGH Cell Surface Binding Assay

C14 cells were equally divided into six-well plates and serumstarved over night. Cells were pretreated in triplicate with varying concentrations of monoclonal antibody or Fab fragment, as indicated, for 30 min at 37 C in 1 ml BB before incubation with 125I-hGH [50,000 cpm (⬃25 pM) per well] either in the presence (to determine nonspecific binding) or absence of 2 ␮g/ml (⬃91 nM) unlabeled hGH for 1 h at 25 C. Cells were washed three times with cold PBS and solubilized in 1 ml 1% SDS-0.1 N NaOH and the lysate was subjected to ␥-counting. Data within each experiment were expressed as specific 125I-GH binding relative to that measured in the absence of antibody pretreatment (PBS control; considered 100%) and displayed in Fig. 6 as pooled data from a number of different experiments, as in the figure legend. Densitometric Analysis Densitometric quantitation of immunoblots was performed using a high-resolution scanner (Epson) and the ImageJ 1.3 program (developed by W. S. Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, MD). In Fig. 7, the degree of PMA-induced receptor loss was estimated as 100% minus the densitometric ratio of mature GHR signal in the presence vs. absence of PMA treatment. This ratio, determined in individual experiments, was displayed in the figure as the pooled data from multiple experiments in the presence of pretreatment with varying concentrations of antibody, as in the figure legend.



Acknowledgments The authors appreciate helpful conversations with Dr. Y. Huang, K. Loesch, J. Cowan, and N. Yang in the Frank Laboratory and the expert assistance of the UAB Multipurpose Arthritis Center Hybridoma Core facility and Dr. M. A. Accavitti-Loper.

12.

13. Received March 9, 2004. Accepted August 24, 2004. Address all correspondence and requests for reprints to: Stuart J. Frank, University of Alabama at Birmingham, 1530 3rd Avenue South, BDB 861, Birmingham, Alabama 352940012. E-mail: [email protected]. This work was supported by National Institutes of Health (NIH) Grant DK58259 (to S.J.F.) and in part by a Veterans Affairs Merit Review grant (to S.J.F.) and NIH Grant DK46395 (to S.J.F.). Parts of this work were presented at the 86th Annual Meeting of The Endocrine Society in New Orleans, LA, 2004.

14.

15.

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