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Endocrinology Vol. 143, No. 1 2-10
Copyright © 2002 by The Endocrine Society

GRH-SOMATOSTATIN-GH

Minireview: Receptor Dimerization in GH and Erythropoietin Action—It Takes Two to Tango, But How?

Stuart J. Frank

Department of Medicine, Division of Endocrinology and Metabolism, and Departments of Cell Biology and Physiology, University of Alabama at Birmingham, and Veterans Affairs Medical Center, Birmingham, Alabama 35294

Address all correspondence and requests for reprints to: Stuart J. Frank, University of Alabama at Birmingham, 1530 Third Avenue South, BDB 861, Birmingham, Alabama 35294-0012. E-mail: frank{at}endo.dom.uab.edu


   Abstract
Top
 Abstract
Introduction
 General Aspects of GH...
 GHR and EpoR...
 Concluding Remarks
 References
 
The receptors for GH and erythropoietin are members of the cytokine receptor superfamily. They are single membrane-spanning proteins that bind ligand in the extracellular domain and couple to cytosolic JAK tyrosine kinases to initiate signaling. The ligand-engaged GH receptor (GHR) and erythropoietin receptor (EpoR) extracellular domains are believed to exist in a dimerized configuration in which a single ligand molecule engages two receptor extracellular domains. The last several years have witnessed a rapid expansion in our knowledge of the structural and functional details of this dimerization process and have forced a reexamination of how the ligand-containing complexes achieve their conformation. For EpoR, there is good evidence that the unliganded receptor is already a preformed dimer that is activated by a ligand-induced change in the receptor conformation. Owing in some measure to the unavailability of the analogous crystal structure of the unliganded GHR extracellular domain, it is still unknown whether GHR adopts a similar preformed dimer/conformational change in response to GH as is found for EpoR. This review critically examines the state of our knowledge pertaining to GHR and EpoR dimerization, noting differences and similarities between the two.


   Introduction
Top
 Abstract
 Introduction
General Aspects of GH...
 GHR and EpoR...
 Concluding Remarks
 References
 
A MAJOR CONCERN of modern molecular endocrinology has been understanding how peptide hormones and growth factors specifically exert their actions. GH is a particularly important growth promoting and metabolic hormone in vertebrates. Though it clearly functions as a classical endocrine hormone, GH (and the related anterior pituitary hormone, PRL) share important structural similarities with a large class of colony-stimulating factors, ILs, and cytokines. Similarly, enough structural and functional relatedness exists between receptors for these hormones and cytokines such that as a group they are referred to as the cytokine receptor superfamily. Despite diversity in the physiology and mechanisms of action of these so-called cytokines and the lack of obvious endocrinologic function of many of them, it is natural for endocrinologists interested in GH action to compare GH receptor activation and signaling to that of other cytokine receptors. Indeed, the rapid pace of progress in the GH/PRL/cytokine field over the past two decades has been fueled in large measure by cross-pollination between various disciplines (endocrinology, hematology, oncology, immunology, etc.) engaged in these studies. This review will address recent developments in our understanding of signaling mechanisms of the GH receptor (GHR) by comparing it with another important cytokine receptor, the erythropoietin (Epo) receptor (EpoR), which is responsible for mediating Epo’s effects on red blood cell production and maintenance. Specifically, by reviewing the similarities and differences in how GH and Epo are thought to trigger their receptors, I will focus mainly on what these systems continue to teach us about the roles of receptor dimerization in hormone action.


   General Aspects of GH and Epo Action and Signaling
Top
 Abstract
 Introduction
 General Aspects of GH...
GHR and EpoR...
 Concluding Remarks
 References
 
GH is produced mainly in the anterior pituitary gland as a 22-kDa polypeptide and is secreted into the circulation under the regulation of GHRH, somatostatin, and Ghrelin (1, 2). Structurally, GH is comprised of four antiparallel helical bundles with loops of differing lengths between the helices (3, 4). Though it can form aggregates, GH circulates and is believed to function largely as a monomer. GH deficiency in humans and in animal models results in diminished postnatal growth that can be largely reversed by GH administration, whereas GH excess results in the syndrome of acromegaly (5, 6). The GHR has a wide tissue distribution but is particularly highly expressed in liver, from which GH elicits substantial production of IGF-1. The role played by IGF-1 in mediating GH’s growth-promoting action will not be discussed here, but recent reports have focused on the relative contributions of GH and IGF-1 in body growth and have highlighted the importance of extrahepatically derived IGF-1 (7, 8). It is clear, however, from elegant clinical descriptions and from knockout mice that generalized absence of the GHR results in the GH insensitivity (Laron) syndrome and that the GHR is the physiologically relevant receptor for GH (9, 10). Epo is produced largely in the adult kidney and in the fetal liver and is the prime regulator of erythropoiesis by promoting survival, proliferation, and differentiation of erythroid progenitor cells. Decrease in oxygen tension induces endogenous Epo production; the Epo then acts distantly (in a hormonal fashion) to increase red cell production (11). Recombinant Epo can be used to treat the anemias that result from end-stage kidney disease and some bone marrow disorders (12).

While each of the cytokine receptor family members are cell surface-expressed transmembrane proteins with extracellular domains that often participate in ligand binding, many of them appear to function only when part of a heterooligomeric assemblage. Some can even function as a subunit of receptors for more than one cytokine (13). For example, the common {gamma} chain is an important component of the receptor complexes for several different ILs (including IL-2, -4, -7, -9, and -15), whereas the common ß chain partners with at least three different {alpha} chains to form the distinct receptors for IL-3, IL-5, and granulocyte-macrophage colony stimulating factor. The GHR and EpoR, unlike these other family members, are each believed to alone (as homodimers in their active signaling conformations) bind their cognate ligands to initiate intracellular signaling. For this reason and because of their particular structural similarities, GHR and EpoR (along with the PRL and thrombopoietin receptors) form a subgroup among the larger group of over twenty Class I cytokine receptors.

Unlike tyrosine kinase growth factor receptors (such as the insulin, IGF-1, and epidermal growth factor receptors) neither GHR nor EpoR encodes enzymatic activity in their cytoplasmic domain. Rather, cytokine receptors physically and functionally couple variably to nonreceptor tyrosine kinases of the Janus family, which includes JAK1, JAK2, JAK3, and TYK2 (13, 14). These proteins are each characterized by the presence of a C-terminal tyrosine kinase domain, an inactive kinase-like (or pseudokinase) domain, and a large N-terminal half believed important for interaction with particular cytokine receptors and other signaling effector and modulator proteins. GHR and EpoR again exhibit similarity in that a wealth of biochemical evidence indicates that both receptors use JAK2 (15, 16, 17). The biological evidence on this point is most clear for EpoR, which unlike the GHR, is itself necessary for in utero development. Targeted disruption of the JAK2 gene results in embryonic lethality due to an absence of red blood cell formation (18, 19).

More detailed attention will be devoted to the activation mechanisms of GHR and EpoR below, but it is important to note that these receptors also share some common activation pathways downstream of JAK2. For example, in comparison to other STATs, both GH and Epo preferentially activate STAT5. However, there may be differences in the degree to which this STAT contributes to GH vs. Epo functions and as to which isoform of STAT5 (A or B) is most relevant for GH and Epo signaling. The STAT5B knockout mouse is small and displays a loss of the normal GH-dependent sexual dimorphism of liver gene expression, suggesting that STAT5B is a particularly important mediator of GH action (20). On the other hand, the STAT5A/B knockout mouse has only a mild anemia (21, 22), suggesting that other pathways may additionally contribute to Epo function. Further, a very recent study examined the effects of reconstitution of mice with EpoR cytoplasmic domain mutants that lacked the tyrosine residues required for STAT5 activation and found again that only a mild anemia resulted (23). It is noteworthy that such reconstitutions of GHR-deficient mice with similarly mutated GHRs have yet to be reported; thus, we do not yet have the full picture of STAT5’s specific biological significance for GH signaling. A number of other pathways known to be important in cell growth, antiapopotosis, and differentiation, such as the MAP kinase and PI3K pathways, are also accessed by both GH and Epo, though we also do not yet know how biologically important are each of these pathways in GH or Epo action.


   GHR and EpoR Dimerization—Similarities and Differences
Top
 Abstract
 Introduction
 General Aspects of GH...
 GHR and EpoR...
Concluding Remarks
 References
 
GHR—preformed or ligand-induced dimer?
As mentioned above, early studies indicated that JAK2 was critical for GH and Epo action (15, 17). It was also observed that physical interaction between the GHR or EpoR and JAK2 could be detected in a variety of assays. Interestingly, most studies have found that receptor-JAK2 interaction is seen even in the absence of ligand and without the requirement for tyrosine phosphorylation of either the receptor or the kinase. For example, we were able to detect specific interaction in vitro between the bacterially expressed GHR cytoplasmic domain and JAK2 (either phosphorylated or nonphosphorylated) extracted from serum-starved mammalian cells (24). This interaction, as found by others for other cytokine receptors, required a membrane-proximal proline-rich element in the GHR referred to as Box 1. Further, we also determined that the GHR-JAK2 interaction, as assessed by coimmunoprecipitation, occurs via an N-terminal region(s) of JAK2 and is detected even if JAK2 is rendered catalytically inactive (25). However, despite this ligand-independent association, various studies have also shown that the degree of interaction between GHR and JAK2 or EpoR and JAK2 is enhanced by treatment with GH or Epo, respectively (15, 17, 24, 26). The basis for this ligand-augmented association is not clearly understood, but it is likely an important observation. It may reflect the ligand’s ability to translate a structural change in the organization or orientation of the receptor extracellular domain to a change in the cytoplasmic domain that allows it to more avidly and productively associate with JAK2.

What might be the nature of these important ligand-induced changes? An elegant series of investigations to address this question began with crystallographic and mutagenetic studies of the GH-GHR interaction in the early 1990s. deVos et al. (4) bacterially expressed the recombinant human GHR extracellular domain (amino acids 1–238 of the 246 predicted extracellular domain residues, referred to as the GH binding protein, or GHBP) and cocrystallized it with recombinant human GH (see Fig. 1AGo). Structurally, the receptor extracellular domain was found to be divided into two ß sandwich subdomains, referred to as subdomain 1 (residues 1–123) and subdomain 2 (residues 128–238) with each subdomain comprised of seven ß strands organized into two antiparallel ß sheets. A four-residue hinge region links subdomains 1 and 2. Surprisingly, the analysis yielded a ligand-receptor complex of 1:2 GH:GHBP stoichiometry. This is particularly intriguing in that GH has no axis of symmetry; yet, two distinct sites ("site 1" and "site 2") in the GH molecule were shown to engage the two GHR extracellular domains at similar contact points on each GHBP. GH contacts the receptor largely at residues in subdomain 1 and the hinge region. The crystal structure showed that the tripartite GH-GHBP2 complex is further stabilized by interaction between the receptor dimer partners via subdomain 2 of each GHBP in a rather extensive region referred to as the dimerization interface. This interface includes six intermolecular bonds between GHBP-I and GHBP-II (serine-145/aspartic acid-152; leucine-146/serine-201; threonine-147/aspartic acid-152; histidine-150/asparagine-143; asparagine-152/tyrosine-200; serine-201/tyrosine-200) and occupies 500 A2, as compared with roughly 1230 A2 and 900 A2 for the site 1-GHBP and site 2-GHBP interactions, respectively. This work was important on many levels, not the least of which is that it represented the first ligand-receptor complex whose structure was resolved to such a high degree. Further, though the structure included no information concerning the transmembrane or cytoplasmic domains, it suggested that the GH-activated GHR was in a homodimeric assemblage and that perhaps the cytoplasmic domain-associated JAK2 molecules were thus approximated as a consequence of GH binding.



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  		Figure 1. GH-engaged GHRs are dimerized. A, Cartoon of the crystallographically determined structure of GH bound to the soluble GHR extracellular domain (GHBP). The structural features of the GHR extracellular domain are indicated, as described in the text. Sites 1 and 2 of GH, which are quite distinct, interact with very similar contact points on each GHBP molecule to result in the GH: GHBP2 complex shown. The extensive inter-GHBP dimerization interface is indicated. This diagram is based on information in Ref. 4 . B, Three possibilities for the GHR’s unliganded conformation. To date, there is no detailed structural characterization of the unliganded GHR extracellular domain. Three possible scenarios discussed in the text are pictured: 1) preformed dimer that binds GH; 2) monomeric GHRs that are dimerized by GH; 3) loosely dimerized GHRs that undergo conformational change in response to GH binding. The drawing for possibility 3 is based on the structure believed to be adopted by the unliganded EpoR (see Fig. 3Go), though no evidence exists for its validity for the GHR.

 
However, there was not (and to date is still not) a description of the structure of the unliganded GHR extracellular domain; thus, it remains unclear whether the receptor: 1) exists as a preformed homodimer before GH binding; 2) is recruited to undergo dimerization by GH; and/or 3) experiences GH-induced conformational changes other than dimerization as well (Fig. 1BGo). The prevailing view has favored the idea that GH promotes GHR dimerization (possibility 2). This is based at least in part on the finding that various measures of GH action in cell culture systems exhibit a bell-shaped GH concentration dependence (Fig. 2AGo). At high GH concentrations, the responses become dose dependently diminished (27). Binding studies and mutagenesis of both GH and GHR extracellular domain have suggested that GH site 1 binding to GHR is of higher affinity than that of site 2 and that binding is thus sequential with site 1 binding to one GHR facilitating site 2 binding with the second GHR (27, 28). The bell-shaped dose response is interpreted to indicate that at very high GH concentration, GH binds to available GHR molecules via site 1 in unproductive monomeric interactions (Fig. 2BGo). A byproduct of the work defining the GHR binding sites 1 and 2 on GH was the further development of GH antagonists (27, 29, 30, 31, 32). Site directed mutation of human GH glycine 120 (mouse GH glycine 119) to arginine or lysine (hGH-G120R or hGH-G120K, respectively) results in a molecule with markedly reduced site 2 affinity (27, 29, 30, 31, 32, 33). When combined with the G120K alteration, mutations that enhance site 1 affinity result in a potent antagonist B2036, which, when PEGylated, has been found efficacious in the treatment of acromegaly (34, 35).



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  		Figure 2. High GH concentrations and GH antagonists inhibit GH signaling. A, Stylized concentration-dependence curves for GH alone and for GH plus GH antagonist treatment of GHR-expressing cells. At very high GH concentration, various biological and biochemical responses (e.g. proliferation, tyrosine phosphorylation, receptor disulfide linkage) become diminished. Addition of GH antagonist (mutant GH with markedly decreased site 2 binding affinity) in the presence of constant (GH) causes inhibition of GH signaling. B, Hypothetical mechanisms of the high-dose suppression of signaling by GH and the GH antagonist effect. For illustrative purposes, the unliganded GHR is shown as a monomer (possibility 2 referred to in Fig. 1BGo) that is dimerized in response to GH (upper panel), though other structures for the unliganded GHR could be adapted to this scheme. High-dose suppression (bell-shaped dose-response curve) for GH (middle panel) is depicted as GH binding to available GHR molecules via site 1 in unproductive monomeric interactions. GH antagonist inhibition of GH signaling is depicted as the antagonist competing for GHR binding via site 1, thus lessening productive GH-induced GHR dimerization. See text for details and for discussion of alternative mechanisms of GH antagonist action.

 
The G120K and G120R have been shown to antagonize GH-induced JAK2 activation and downstream tyrosine phosphorylation in a number of cellular model systems (26, 36). A straightforward explanation as to their inhibitory mechanism is that the antagonists compete with GH by binding to the GHR via site 1 but do not allow GHR dimerization because of the mutation in site 2, thus lessening productive GH-induced GHR dimerization (Fig. 2BGo). Thus, the functional findings with GH antagonists could support the view that GH normally works to promote GHR activation by the sequential dimerization of previously undimerized GHRs, as above. However, they could equally well be consistent with the presence of rather loosely associated, yet dimerized, GHRs in the basal state that become a more avidly associated dimer in the presence of GH. In part to address this issue, two independent groups have generated crystal structures of a GH antagonist bound to the GHBP (37, 38). In both cases, the crystals revealed a 1:1 stoichiometry of GH antagonist:GHBP with GH antagonist site 1 mediating the association with the receptor, perhaps favoring (but not directly testing) the notion that the unliganded GHR is a monomer. In these structures, the conformation adopted by the bound receptor did not significantly differ from that of the site 1-bound GHBP in the case of the wild-type GH:GHBP2 structure. Thus, it was concluded by these investigators that site 2 binding of GH to a second GHR did not induce significant conformational change in the already (site 1) bound GHR.

Yet, the situation as it pertains to the cell surface GHR may prove more complex than that inferred from the crystal structures of the recombinant hormone-GHBP complexes. For example, studies of the effects of treatment of GHR-expressing cells with the GH antagonist have yielded both expected and surprising results. As might be predicted, several groups have observed that treatment of GH-responsive cells with the GH antagonist by itself fails to promote proliferation or tyrosine phosphorylation of the GHR and other cellular proteins and that coaddition of GH antagonist with GH at ratios of 3:1–5:1 inhibits these GH-induced effects (26, 27, 32, 36, 40, 41). But surprisingly, chemical cross-linking studies showed that radiolabeled G120R interacts with cell surface GHRs so as to be in a complex that includes a dimeric GHR (40) in contrast to the crystallographic studies of the antagonist-GHBP interaction. Further, rather than interacting with surface GHRs in a fashion that prevents any aspect of receptor function, two groups have determined that both the G120R and B2036 GH antagonists cause the GHR to internalize with kinetics indistinguishable from those found with wild-type GH treatment (40, 41). These findings suggest that the GH antagonists, though they clearly inhibit the effects of GH and by themselves are not GH agonists, can indeed either cause some degree of GHR dimerization and/or may be interacting in some way with an already-existing GHR dimer (42).

Our own findings add another aspect to the GHR dimerization issue. Using anti-GHR immunoblotting, we have observed in several cell lines that GH induces formation of a covalent GHR dimer detectable only when the receptor is electrophoretically resolved under nonreducing conditions (26, 43, 44, 45). This disulfide-linked GHR form does not appear to result from disulfide exchange following protein extraction and it is abrogated by mutation of cysteine-241 (the only unpaired GHR extracellular cysteine residue). This cysteine is highly conserved among species and is predicted to reside at the GHR juxtamembrane stem. We are as yet uncertain of its role, but our preliminary evidence suggests that the disulfide-linked GHR may be trafficked differently in response to GH than those GHR dimers (roughly one-half by our estimate) that do not undergo this linkage (44 and unpublished results). Independent of its exact function, the disulfide-linked GHR is not formed in response to G120K and G120K antagonizes its formation by GH (26). We currently view the disulfide linkage as a biochemical proxy for "proper" GH-induced GHR dimerization and note that unliganded GHRs are not disulfide linked.

Does GH induce GHR conformational change?
These observations do not yet allow resolution of the issue of whether the GHR exists as a dimer of some sort before engagement by GH, but they do suggest that if it is a preformed dimer, GHR likely undergoes a GH-induced conformational change to achieve the status required to trigger receptor signaling. Indeed, there is emerging evidence favoring the possibility of such a conformational change. Mellado et al. (46) showed that a monoclonal antibody raised against the GHR extracellular domain and reactive with the hinge region between subdomains 1 and 2 was capable independent of GH of promoting GHR-mediated activation of intracellular tyrosine phosphorylation and cellular proliferation. Moreover, GH (but not G120R) treatment enhanced the recognition of the GHR epitope by that antibody. Rowlinson et al. (47) similarly found that a stimulatory monoclonal antibody that causes GHR dimerization lost its ability to stimulate when the F'-G' loop of subdomain 2 of the receptor extracellular domain was mutated. This region of the receptor was previously implicated by the same group as possibly undergoing a GH-induced conformational change (48). Collectively, the findings of these two groups may suggest that a GH-induced conformational change in the GHR extracellular domain, along with GHR dimerization, may be needed for optimal receptor activation. One interesting corollary to this interpretation might be that our observed GH-induced disulfide linkage of receptors may only be allowed when the GH-induced conformational change is achieved. Such a possibility is testable and could yield mechanistically important information about early steps in GHR activation.

EpoR—a different kind of dimer?
Given the relatedness of the GHR and EpoR in the cytokine receptor superfamily and the degree to which there are similarities in their signaling mechanisms, it is worth comparing the two receptors with regard to the role that dimerization plays in activation. Work in the early 1990s, before a structural understanding of the GH-GHBP complex, indicated that a mutant murine EpoR with a change in the extracellular domain arginine residue 129 to cysteine (R129C) conferred Epo-independent proliferation in factor-dependent cells (49). Primary sequence comparison later suggested that arginine-129 was in a position in the EpoR extracellular domain analogous to the then recently defined dimerization interface in the GHR extracellular subdomain 2 in the GH-GHBP2 crystal structure. This result prompted biochemical analysis of the R129C EpoR mutant and it was found to exist in a constitutive (Epo-independent) disulfide-linked dimer when expressed in cells (50).

This finding suggested that EpoR activation, like GHR activation, might normally be mediated by Epo-induced EpoR dimerization of some sort, which was being mimicked in an Epo-independent fashion by the presence of the cysteine-mediated disulfide linkage in the case of the R129C mutant. (Notably, in distinction to our findings in several species that GH induces disulfide linkage of the GHR, Epo-induced EpoR disulfide linkage has not been observed for the wild-type EpoR.) Further evidence for the formation of a Epo:EpoR 1:2 complex akin to that for GH-GHR came from biophysical studies of the interaction of recombinant Epo and EpoR extracellular domain. These studies indicated that two Epo binding sites interacted with EpoR extracellular domain monomers and that the high affinity site (analogous to GH site 1) was of far greater (1000-fold) affinity than that of the low affinity (site 2) site (51). This difference in the affinities of the two Epo sites was substantially greater than that estimated for the analogous GH sites, suggesting that, though similar, there might be important differences in the mechanisms of inducible EpoR vs. GHR dimerization. Along these lines, it is interesting to note that far greater concentrations of Epo relative to GH are apparently required to cause self-antagonism (the bell-shaped concentration dependence) of signaling through the EpoR than are required for GH self-antagonism (52).

In the past several years, more detailed structural information about the Epo-EpoR association has emerged. The first crystal structures of the liganded recombinant EpoR extracellular domain (EBP) probed its interactions with Epo-mimetic and Epo-antagonistic peptides (53, 54). These peptides with structures unrelated to Epo were originally isolated by phage display screening procedures designed to obtain Epo-mimetics (55). In each case, a dimeric ligand (mimetic or antagonist) engaged an EBP dimer, but the orientation between the EBP dimer partners differed in the case of the mimetic- vs. antagonist-bound EBPs. This difference in orientation was interpreted as related to whether the intracellular domains of the EpoR would be brought into productive vs. unproductive signaling orientations. Interestingly, contacts between the EBP dimer partners in both cases were much less extensive than those found in the GHBP dimer in complex with GH.

Owing to difficulty in crystallizing Epo, the Epo-EBP interaction was not studied until late 1998 (56); in fact, this structure could only be obtained by making selected mutations in both Epo and EBP. The Epo mutein, not surprisingly, adopted an overall antiparallel helical bundle configuration similar to GH. The Epo-EBP complex, like the GH-GHBP complex, assumed a 1:2 stoichiometry with "site 1" and "site 2" Epo sites interacting with each EBP. However, in sharp distinction to the GHBP dimer, the EBP dimer found in complex with Epo (like that complexed with the Epo-mimetic and the Epo-antagonist) evidenced a relatively minimal dimerization interface. Interestingly, the only interaction in the EpoR dimerization interface is between the side chains of residues serine-135 and glutamic acid-134 on adjacent EPBs (56), a position in the vicinity of the R129C mutation that confers constitutive activation. Thus, the crystallographic and kinetic analyses of the ligand-engaged EpoR extracellular domain dimerization allow several potentially important distinctions to be drawn between EpoR and GHR dimerization: 1) EpoR displays a markedly lower sensitivity than GHR to high-dose suppression by its ligand; 2) EpoR extracellular domain, unlike GHR, adopts a dimeric conformation even when engaged by a functional Epo-antagonist; 3) the dimerization interface in subdomain 2 of the Epo-engaged EBP is far less extensive than is that of the GH-engaged GHBP, despite the adoption of a 1:2 ligand:BP assemblage by both.

Whether these rather surprising distinctions are meaningful is as yet uncertain. However, recent structural and mutagenetic studies of the EpoR over the past 3 yr have produced very interesting results that should make further comparison with the GHR more compelling. Companion reports by Livnah et al. and Remy et al. in 1999 (57, 58) yielded important evidence that the EpoR in the unliganded state exists as a dimer. The crystal structure shows that the unliganded EBP dimer is in an open-scissors-like configuration with the dimerization interface consisting of self-association of the two ligand binding sites on the EBPs and with the C-terminal end of the subdomain 2 regions of the EBPs being quite far apart (over 70 Å) (57) (Fig. 3Go). In the liganded EBP structures, these C-terminal regions become much closer (30 Å for the Epo-engaged EpoR). Thus, it is envisioned that the preformed dimer, by keeping the cytoplasmic domains apart, is in an inactivated state, but ligand occupancy brings the extracellular and cytoplasmic domains into proximity and allows signaling. Fragment complementation assays (58) confirmed these data by demonstrating a dramatic ligand-induced enhancement of proximity of the cytoplasmic domains of EpoR dimers. Thus, these studies strongly point to the existence of preformed EpoR dimers that are activated by a distinct conformational change in response to ligand.



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  		Figure 3. Unliganded EpoR is a dimer that undergoes dramatic conformational change in response to Epo. As described in the text, this schematic combines crystallographic data regarding the EpoR extracellular domain (from Refs. 56 57 ) and fragment complementation data (from Ref. 58 ) to indicate that the extracellular domains of EpoR are likely dimerized in an "open scissors-like" conformation (left side) that undergoes a dramatic conformational change in response to Epo (right side). This Epo-induced conformational change in the EpoR extracellular domain is believed to be translated to the cytoplasmic domains and their associated JAK2 tyrosine kinases, allowing closer JAK2 proximity and facilitating transphosphorylation and activation. The structure of the EpoR cytoplasmic domain and the structural details of association of the cytoplasmic domain with JAK2 are unknown. The diagram is based in information from Refs. 56 57 58 .

 
Is there dimer/conformational change in the EpoR mediated by one or more regions other than the extracellular domain? This question has received attention within the last several months, but a definitive answer remains yet elusive. Two groups have shown that the transmembrane domain of the EpoR is endowed with powerful and specific self-oligomerizing ability, but the groups differ as to the consequences of this capacity. Constantinescu et al. (59), using a copatching technique to study epitope-tagged full-length EpoRs at the surface of living cells, concluded that the transmembrane domain interaction among unliganded EpoRs maintains them in an inactive state and that EpoR-activating ligands work by causing a conformational change and an undoing of this inhibitory dimerization. Kubatzky et al. (60) also found strong and specific self interaction between EpoR transmembrane domains but concluded that abrogation of this dimerization capacity by mutagenesis impaired signaling via full-length EpoRs in erythroid progenitor cells. Thus, this group’s conclusion is that transmembrane dimerization may drive, rather than inhibit, signaling. These observations that the transmembrane domain may serve as an important dimerization interface for the EpoR are compelling, but further studies will be required to more precisely indicate the mechanism of this region’s contribution to EpoR triggering and to determine whether the GHR or other cytokine receptor transmembrane domains can serve a similar role(s) in the activation of those receptors.

Finally, another recent study highlighted the likely structural importance of the EpoR perimembraneous cytoplasmic domain in facilitating transmission of the presumed conformational change in the receptor’s extracellular domain to productive interaction of the cytoplasmic domain with JAK2. Using a combination of computer modeling and mutagenesis, Constantinescu et al. (61) demonstrated that a juxtamembraneous cytoplasmic domain element just N-terminal to the EpoR Box 1 element contains a hydrophobic motif necessary for JAK2 activation and receptor signaling. This motif is likely to be conformationally rigid and {alpha}-helical. Also, mutations of critical hydrophobic residues within it abrogate Epo-induced JAK2 activation. However, insertional mutations that alter the register of the {alpha}-helix but leave intact the hydrophobic residues, result in Epo-induced JAK2 activation without EpoR tyrosine phosphorylation or downstream signaling. Thus, it was proposed that the juxtamembrane/Box 1 region is important in both activation of JAK2 and positioning of the EpoR cytoplasmic domain in the conformation to be a proper JAK2 substrate. These authors note that this juxtamembraneous motif is highly conserved among cytokine receptors including the GHR. It may thus also be a fundamentally important region for coupling GH occupancy to GHR-JAK2 functional association.


   Concluding Remarks
Top
 Abstract
 Introduction
 General Aspects of GH...
 GHR and EpoR...
 Concluding Remarks
References
 
The GHR and EpoR are among the most intensively studied cytokine receptors with regard to molecular mechanisms of their activation. It is now clear that dimerization is quite important in activation of both receptors, though the specifics of the dimerization interfaces and the degree to which the unliganded receptors exist as dimers may differ substantially between the GHR and EpoR. Several physiologically relevant aspects of signaling via the GHR and EpoR will undoubtedly become better understood as details of the dimerization of these receptors are clarified. The recent finding that an activating nonEpo-mimetic peptide activates the EpoR by binding to it at a site distinct from the Epo binding site exemplifies a pharmacologically important development in this area (62). Similarly, better understanding of the nature of the GHR dimer will allow a firmer grasp of aspects of GHR function. For example, there is now data indicating that placental lacotgen, a distinct GH gene family member, causes functionally competent heterodimerization between the GHR and PRL receptor (63). How does this occur? And how is it that GH, but not the GH antagonist G120K, can inhibit the inducible proteolysis of the full-length GHR and shedding of the GH binding protein in an apparently dimerization-dependent fashion (45). It will be interesting to gain insight into these and other issues of cytokine signaling as we refine our knowledge of the mechanisms and implications of cytokine receptor dimerization.


   Acknowledgments
 
The author acknowledges helpful discussions with Drs. K. Harris, P. Ney, R. J. M. Ross, P. Sanders, and M. J. Waters and critical review of the manuscript by Dr. J. J. Kopchick.


   Footnotes
 
The studies carried out in the author’s laboratory were supported in part by NIH Grants DK-46395 and DK-58259 and a VA Merit Review award.

Abbreviations: EBP, EpoR extracellular domain; Epo, erythropoietin; EpoR, Epo receptor; GHBP, GH binding protein; GHR, GH receptor.

Received August 22, 2001.

Accepted for publication October 2, 2001.


   References
Top
 Abstract
 Introduction
 General Aspects of GH...
 GHR and EpoR...
 Concluding Remarks
 References
 

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