Structural and functional aspects of the interaction between growth hormone and its receptor

1

Biochimica et Biophysica Acta, 1203 (1993) 1-10 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4838/93/$06.00

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BBAPRO 34601

Structural and functional aspects of the interaction between growth hormone and its receptor James Beattie Hannah Research Institute, Ayr (UK) (Received 16 March 1993)

Key words: Growth hormone; Growth hormone receptor; Crystallography; Mutant; Cytokine

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

II. Structural aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. X-ray crystallography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Studies with mutated GH molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Immunological studies of the GH receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Biological activities at high GH concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. GH receptor in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Wider implications: the cytokine receptor superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. Introduction

Growth hormone (GH) is a polypeptide hormone secreted principally by somatotroph cells of the anterior pituitary. The biological activities displayed by the hormone are diverse and include somatogenic (growth promoting), lactogenic, insulin-like and diabetogenic effects [1]. In addition, GH is thought to play a role in the conversion of pre-adipocytes to the fully differentiated adipose cell [2].

Correspondence to: J. Beattie, Hannah Research Institute, Ayr KA6 5HL, UK. Fax: + 44 292 671052. Abbreviations: GH, growth hormone; GHR, growth hormone receptor; GHBP, growth hormone binding protein; IL, interleukin; PRL, prolactin; G-CSF, granulocyte colony-stimulating factor; EPO, erythropoietin; GM-CSF, granuloeyte macrophage colony-stimulating factor.

Like other polypeptide hormones, growth hormone exerts its effect by binding to a specific, high-affinity cell surface protein; the growth hormone receptor (GHR). Studies on GH and GHR at the molecular level have been aided by the fact that the sequences of both hormone and receptor are available for many species (see, for example, Refs. 3,4). In the case of GH, this has ensured sufficient material (through recombinant DNA technology) to allow structural characterisation of the protein. Similarly, the availability of cloned GHR sequences in suitable expression vectors has allowed the creation of stably transfected cell lines expressing all or partial sequences of the GHR at the cell surface. These in turn allow detailed studies on the structure of GH-GHR interaction and on subsequent signalling mechanisms. In this review, I shall discuss some of the structural features of GH and GHR and of the unusual complex formed between the two proteins. A unifying theme will be how these structural and

Fig. 1. (A), Representation of the crystal complex formed between hGH and two molecules of hGHbp. The four a helices of the GH molecule are shown in yellow (numbered 1-4 with connecting loop structures coloured red). For hGH bp 1 loop structure is shown in green and for bp 2 in blue./3-strand structure in both binding proteins is brown. Less well-defined areas of structure are represented by dotted lines. (B), Identification within a ribbon structure diagram of GH of those areas of the hormone at the interface of hormone-bp 1 (site I, green) and hormone-bp 2 (site II, blue). Both figures are taken from Ref. 6.

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100

1000

104

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.... ~' . . . . . . . . 1000 104

hGH variant [nM}

Fig. 2. (A), Stimulation of [3H]thymidine uptake into FDC-P1 cells expressing the hGH-mG-CSF hybrid receptor. (e), wt-hGH; (It), G120R-hGH. (B), Inhibition by G120R (e) of wt-hGH (1 nM) stimulated [3H]thymidine incorporation. Both Fig. 2A and B are adapted from Ref. 8.

chemical observations relate to the biology of GH both in vitro and in vivo. Finally, some of the wider implications of the findings in G H / G H R research for the cytokine superfamily of receptors/ligands will be discussed. II. Structural aspects

II-A. X-ray crystallography A major advance in the field of GH research came with the publication of the X-ray crystal structure of porcine (p) GH [5]. Inspection of this structure revealed the protein to be folded as a four a-helix bundle with an unusual up-up-down-down topography. Connections between the helices are formed by pieces of loop/coil structure of varying lengths, leaving a small portion of less well-ordered structure at both the Nand the C-terminus of the molecule. This was followed by a landmark study which described the crystal structure of the complex formed between human (h) GH and the recombinantly produced hGH binding protein [6] (see Fig. 1A). The salient features of this unusual structure include the assymetric binding of hormone to two molecules of BP, such that there are spatially distinct sites on the hormone (sites I and II) interacting with essentially the same region on each BP. At these hormone-BP interfaces, therefore, there are defined residues on GH which form binding 'epitopes' (see Fig. 1B). These sites are largely discontinuous (conformational) in character and are derived from both helix and loop/coil regions of GH [6,7] (see Fig. 1B). A third inter-molecular interface is defined between the GHBPs; a putative 'dimerisation domain'. In addition to the crystallographic evidence of a 1 : 2 binding stoichiometry between hormone and BP, interaction between the two proteins in solution phase also occurs at a binding stoichiometry of 1:2 [7]. Further to this, it

seems that binding of hGH occurs in an ordered, sequential fashion with interaction firstly via site I on hGH with a single BP molecule followed by completion of complex formation with site II on hGH binding to a second BP [8]. As noted above, crystallisation and physico-chemical studies of GH-GHBP interactions were performed with recombinantly produced GHBP. As there is very strong agreement that this high-affinity soluble GHBP represents the extracellular domain of the membrane-bound growth hormone receptor (GHR) in humans and other species (reviewed in Ref. 9), speculation arose as to the possible role of receptor dimerisation in the biological activity of GH. There are three current lines of evidence which support the hypothesis that receptor dimerisation is required for biological activity of the hormone; studies with site-directed mutants of the GH molecule, studies with anti-receptor antibodies and limited observations of biological responses at high GH concentrations.

II-B. Studies with mutated GH molecules Perhaps one of the strongest and most elegant pieces of evidence in support of the necessity of receptor dimerisation for biological activity is from the same group which described the X-ray crystallographic structure and additional physico-chemical parameters of GH-GHBP interaction [6-8,10,11]. Using the myeloid leukaemia cell line FDC-P1 transfected with a construct which contains the extracellular domain of the GHR linked to the transmembrane and intracellular domain of GCSFR (granulocyte colony-stimulating factor receptor), these workers demonstrated a stimulation of thymidine incorporation in response to wild-type (wt) hGH. In contrast the site-directed mutant G120RhGH (where the native glycine (G) residue at position 120 is replaced by an arginine (R) residue) is inactive

and furthermore inhibits the activity of wt-hGH (see Fig. 2). These findings are explained by the observation that G120 is in the region of site II on hGH. Thus, whilst G120R can interact with a single receptor molecule through site I on the hormone (which is borne out by identical reported K d values for wt-hGH and G120R-hGH) it is unable to bring about receptor dimerisation, arguing that in this instance dimerisation is required for biological activity. These findings corroborated earlier work using an in vivo model, where mice made transgenic with the bovine (b) GH mutant G119R (the same mutation as that described above for hGH) demonstrated a dwarf phenotype [12]. Again, bGH-Gll9R showed the same affinity for mouse liver GHR as wt-bGH and the inference was drawn that Gll9R-bGH present in transgenic mouse serum inhibited the activity of endogenous mouse GH. If it is assumed that the tertiary structure of bovine GH [13] is similar to that described for porcine (p) [5] and hGH [6] and binding occurs in a similar stoichiometric and sequential fashion, then non-dimerisation of receptor in vivo by Gll9R-bGH provides an explanation for these observations. Using a different site-II mutant bGH (El17L-Gl19R-A122D-bGH), the same group [14] demonstrated a failure of the hormone to differentiate the 3T3-F442A pre-adipocyte cell line. In addition this mutant did not cause the insulin-like or lipolytic effects normally associated with GH action in primary cultures of rat adipocytes and furthermore antagonised the activity of wt-bGH in this system. These data indicate that receptor dimerisation is probably required for each of these three separate biological activities. Early studies had suggested that site-II mutants would show deficiencies in more than one biological activity. A particularly interesting example is in relation to des 1-13 hGH, which completely lacks some of the residues involved in site-II binding. This molecule inhibits the ovine prolactin ( o P R L ) / h G H stimulation of fat synthesis and a-lactalbumin secretion by lactating bovine mammary gland explants, as well as inhibiting receptor down-regulation in vivo and in vitro [15,16]. As all these activities are mediated through the prolactin receptor, it suggests indirectly that receptor dimerisation of a type similar to that seen for GH may be required for prolactin signalling. Further studies, however, are required to establish this and indeed molecular dissection of PRL-receptor interactions are already underway [11]. There are some reports in the literature on the activity of GH mutants which are difficult to reconcile with the structural and mechanistic data available on GH-receptor interaction. For example, Becker and Shaar [17] have reported that the site-II deletion mutant des 1-8, 135-145 hGH has little insulin-like or diabetogenic activity but retains 70% of its somato-

genic activity. This suggested that the different biological effects of GH may be physically separated within the structure of the molecule, an idea which had been suggested previously [18,19]. That this is a controversial area, however, is indicated by the fact that a recent report [20] demonstrated that a similar GH mutant, des 1-7 hGH had little somatogenic or insulin-like activity. Studies with site-I GH mutants are less extensive. The sequential model for GH interaction with receptor would predict that GHs with major structural alterations of site I would not bind to the GHR and would therefore be biologically inactive. Indeed, the double site-I mutant K172A/F176A demonstrates an approx. 300-fold decrease in affinity for the recombinantly-derived hGH binding protein and a correspondingly reduced activity in an in vitro bioassay [8]. Similarly, Uchida et al. [21], using a series of hGH site-I mutants, P59A, P61A, P59A-P61A and za62-67, demonstrated that the ability of the four mutants to cause adipose conversion of 3T3-F442A cells paralleled their affinities in a radio-receptor assay. To date, therefore, the data generated on site-I and site-II GH mutants are qualitatively and quantitatively different. For the existing site-II mutants, the response appears to be 'all-ornothing' with the hormone being inactive both in vitro and in vivo and this inactivity being associated with non-dimerisation of the receptor. For site-I mutants a quantitative aspect pertains to the data with activity mirroring the affinity of interaction of hormone with receptor. The evidence suggests that for site-I mutants there is no obvious defect in receptor dimerisation. An interesting situation pertains in the case of the 20-kDa GH species. This pituitary-derived natural variant of GH represents 5-10% of circulating hormone and indeed is the most abundant naturally occurring isoform of the hormone [19,22]. The 20-kDa GH is formed by alternative splicing of mature GH, such that residues 32-46 of the hormone are removed. This deletion removes a portion of the GH molecule which is believed to contribute to binding site I. As such it might be expected that 20-kDa GH may demonstrate an attenuation of some or all of its biological activities. However, conflicting data have been reported with this hormone. Firstly, using molecular biology techniques, Cunningham et al. [7] have generated the mutant hGH z132-46, equivalent to the circulating 20-kDa form of hGH. They report that this mutant is as effective as wt hGH in causing dimerisation of BP in solution and is equipotent with wt hGH in an vitro bioassay using the stably transfected FDC-P1 cell line. In biological studies with 20-kDa hGH, it was reported that the hormone possessed full growth-promoting activity but weak insulin-like activity [23]. This finding was rationalised on the basis that 20-kDa hGH binds poorly to rat adipocytes (insulin-like effects) but normally to IM-9

cells (somatogenic/growth promoting effects), although Waters et al. [24] reported no difference in affinities between native 22-kDa and 20-kDa hGH in binding to rat liver or adipose membranes. A subsequent report has observed reduced affinity of 20-kDa hGH for human liver receptor and serum BP and indeed evidence has been presented for a low-affinity 20-kDa-specific GH binding protein in human serum [25]. Whether such a receptor protein exists in cell membranes requires further investigation.

II-C. Immunological studies of the GH receptor The observation that a panel of monoclonal antibodies (MAbs) to the GHR were biological agonists of GH action, whereas their Fab fragments were inactive argues that antibody bivalency and by inference receptor dimerisation is required for signal transduction [8]. The argument that this may simply reflect affinity/avidity differences between MAbs and their corresponding Fab fragments is countered by the fact that Fab fragments binding at, or close to, the site of GH-GHR binding are able to antagonise the biological activity of GH [8]. In addition, it has been reported that one MAb is able to prevent receptor dimerisation (presumably by binding at, or in the vicinity, of the interface between the two GHBPs). The demonstration that this antibody is biologically inactive further argues for the necessity of receptor dimerisation for biological activity. Similar observations have been made recently with MAbs and Fab fragments to the rat prolactin receptor [26], arguing that the phenomenon of dimerisation may be a common feature in this receptor superfamily (see later).

II-D. Biological activity at high GH concentrations There is, therefore, a considerable body of evidence which supports the hypothesis of GHR dimerisation as a pre-requisite for biological activity. If the sequential mechanism of GH binding to receptor (site I then site II) is also valid, then the prediction may be made that at high GH concentrations some attenuation of hormone activity should be evident. This follows because at high G H / G H R ratios increasing proportions of GHR would be occupied univalently through site-I binding to GH. Subsequently, fewer unoccupied GHRs would be available for dimerisation through site II on GH. This hypothesis was formulated and tested by Fuh and her co-workers [8] and indeed at high GH concentrations (1-10 ~M), thymidine uptake into the G H R / GC-SFR construct transfected FDP-C1 cell line decreased (see Fig. 2A). Support for this view has been provided by a very recent study [27] which has examined the hGH induced phosphorylation of cellular proteins in the human lymphocyte cell line IM-9. This cell line responded to hGH by increasing the level of

tyrosine phosphorylation of two proteins of 93 and 120 kDa. This effect was attenuated by co-incubation with the site-II mutant G120R, confirming the reported antagonistic effect of this molecule. However, examination of a dose-response curve for hGH-stimulated tyrosine phosphorylation indicated inhibition at high concentrations of hGH. Thus, whereas at 100 nM the effect of hormone appeared to be maximal, a 10-fold increase in hGH concentration to 1 /~M resulted in a dramatic decrease in the level of phosphorylation of the two proteins. Whether this inhibitory effect of GH at high concentrations has any physiological relevance remains to be determined. IlL GH receptor in vivo

The extensive structural and functional data now available on GH and GHR, obtained by methods outlined above, allow a retrospective analysis of those studies which have aimed to define the structure of GH-GHR complexes in various tissues from different animal species. At the forefront of the methodologies used has been the covalent affinity cross-linking of 125I-labelled GH to membranes prepared from various tissues and the subsequent examination of the radioactive complexes formed by SDS-PAGE and autoradiography. Studies of this nature have been reported in membranes derived from rat adipocytes [28], rat hepatocytes [29,30], sheep liver [31], human liver [32], human lymphocytes [33], mouse liver [34], mouse fibroblasts [35-37] and in Chinese hamster ovary (CHO) cells expressing the transfected ovine (o) GH receptor [38]. For each of the species mentioned above, specifically labelled receptor was detected, with the exact molecular mass of the complexes dependent on the tissues studied. Despite this, there were some common patterns observed in these data. Firstly, under non-reducing conditions, two main bands were almost always labelled and the higher molecular mass band was approximately twice the size of the lower (see Table I). Secondly, in some cases the presence of reductant during SDS-PAGE analysis resulted in the disappearance of the high molecular mass complex and an apparent increase in intensity of the lower molecular mass complex [28,29,31,32] and indeed by reducing/ non-reducing 2-dimensional gel-electrophoresis autoradiography, it has been shown that the component(s) in the lower molecular mass complex are present also in the higher molecular mass complex. Relating these observations to the previously described crystal structure of the GH-GHBP complex it is attractive to suggest that GH receptor monomers may be disulphidebonded by an inter-molecular bond at the free cysteine residue close to the transmembrane domain of the receptor. It should be emphasised, however, that convincing experimental evidence for the existence of such

6 TABLE I

Molecular mass by affinity cross-linking of GHRs from various sources Note: the molecular mass of G H (22 kDa) has not been subtracted from reported values. Species/Tissue

Rat adipocyte Rat hepatocytes

Sheep liver H u m a n liver H u m a n lymphocytes (IM-9) Mouse liver Mouse fibroblasts

C H O cells (transfected with o G H R )

Molecular mass ( × 1 0 -3 ) (kDa) 270 134 280 200 100 86 43 140 75 124 75 270 140 125 62 300 230 130 60 255 134 239 134 170 95

References

28 29

30 31 32 33 34 35 a

36 a,b

37 a,b 38

a 3T3-F442A and 3T3-C2 cells. b Variable a m o u n t s of higher molecular mass complexes also reported.

a complex is at present lacking. However, the presence of disulphide-linked homodimers of GHR has been suggested previously [25,33] and interestingly this free cysteine residue (at position 241) lies in the region of the BP-BP interface. It should be noted though that the site-directed mutant of GHR-C241R (replacing this native cysteine with arginine) is active when transfected into the FDC-P1 cell line [8], arguing that although disulphide bond formation between receptors may occur in vivo it is not a pre-requisite for a biologically active signal-transducing complex. Although these considerations provide a ready synthesis of the affinity-cross linking and structural data, there remain a number of inconsistencies which require examination. (i) If the stoichiometry of GH-GHR interaction is indeed 1:2, then why are lower molecular mass (presumably monomeric GH-GHR complexes) detected at all? There may be a number of possible explanations for this. Firstly, consistent with the idea of the sequential mechanism of GH binding to GHR is that GH may be present in excess over GHR in the cross-linking

reaction and, therefore, 1:1 stoichiometric complexes between hormone and receptor are seen. In most affinity cross-linking studies the concentration of [125I]GH employed is reported and usually falls in the range 0.5-5 nM (see Refs. 28-38 and Table I). However, calculating the concentration of available GHR in a particular preparation is less straightforward. In these instances assumptions may have to be made about the number of receptors per cell a n d / o r the abundance of GHR in a particular membrane preparation. For example, one report estimates the number of GHRs on rat adipocytes at 20000 copies per cell [39]. In 107 cells therefore (a typical number for cross-linking experiments), the concentration of GHR would be approx. 0.33 nM, making the [~25I]GH present approx. 10-fold in excess of receptor. Under these conditions therefore, univalent hormone-receptor complexes may be formed. Nevertheless, these simple calculations reveal how the types of hormone-receptor complexes obtained may be crucially dependent on the number of receptors/cell (which is often not known), the number of cells per cross-linking reaction and the concentration of [125I]GH. These considerations are often overlooked by researchers. Secondly, inefficient crosslinking of [125I]GH at either site I or site II would lead to appearance of univalent hormone-receptor complexes. Finally, the presence of endogenous GH in the membrane preparation would have the effect of increasing the hormone/receptor ratio and may bias the cross-linking reaction towards the production of univalent complexes. For this reason, it is probably advisable to include a high concentration salt wash of membrane preparations prior to affinity cross-linking. (ii) The second observation from affinity crosslinking experiments which requires some comment is the shift seen in the higher molecular mass labelled band following SDS-PAGE autoradiography under reducing conditions. If indeed GH is covalently linked to two molecules of binding protein during the cross-linking reaction, then even under reducing conditions the 1 : 2 stoichiometry of the hormone : receptor complex should be retained (see Fig. 2). Under these conditions no dramatic differences should be seen in the positions of labelled bands, irrespective of whether gels are run under reducing or non-reducing conditions. However, as indicated above several labs have reported that this is not the case. As outlined above, one possible superficial explanation of this data may be that cross-linking does not occur efficiently at site I or site II, such that under the denaturing conditions used in SDS-PAGE, non-covalently-bound receptor proteins are separated and only the monovalent receptor/[125I]GH complex is visualised. Another possibility may be that in the hormone receptor complex, the GH molecule is cleaved; under these circumstances reduction of the two intra-molecu-

lar disulphide bonds in GH and inter-molecular bonds between the two receptor proteins would lead to two receptor molecules with an 125I-labelled GH fragment bound. It should be noted that the existence of cleaved forms of GH has been known for some time [40] but have to date largely been thought to be produced artefactually during extraction of pituitary glands or on storage of the hormone. Nonetheless, cleavage of various GHs by membrane fractions derived from rabbit liver [41], rat kidney [42] and rat liver [43] has been demonstrated and very recent evidence has been provided to suggest that this in situ cleavage of GH may be of physiological significance [44]. As the above mentioned membrane fractions are often used in affinity cross-linking experiments it seems possible that proteolysis of the hormone may occur in these preparations. Some of these possibilities have been represented schematically in Fig. 3. I would once again emphasise, however, that to date there is only limited and indirect evidence for disulphide-linked G H R homodimers.

Finally in this section, examination of some of the data in Table I indicates that some labs have reported affinity cross-linked complexes larger than could be accounted for by a 1:2 stoichiometry of binding between hormone and receptor [36,37] and indeed some smaller than the molecular mass of G H R calculated from the cloned protein from various species. In terms of the higher molecular mass complexes, it is possible that the free cysteines on the G H R may not be involved in the formation of homodimers of the receptor but rather may be disulphide linked to other protein(s) in the vicinity. Interestingly, some evidence has been presented for the association of protein(s) with the G H R which may be involved in signal transduction through this receptor [45]. The lower molecular mass complexes reported, for example for human liver (75 kDa [32]), mouse liver (62 kDa [34]) give a molecular mass for the receptor (subtracting 22 kDa for GH) of 53 and 40 kDa, respectively. As the cloned GHRs of different species suggest a protein comprising approx. 620 residues (reviewed by Matthews [46]), these low

b)

GHR(110k)

(242k)

(1.32k)

GH (22k) Covalent linkage of hormone to R.

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proteolytic cleavage of GH.

2

2

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o)

(242k)

(242k)

C> SH HS

(121k)

(242k)

1

I

2

t"t

I

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(121k) 2

I

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Fig. 3. Highly schematic diagram of putative complexes formed between GH and GHR and subsequent behaviour during reductive S D S / PAGE/autoradiographic analysis. In (a) GH is covalently cross-linked to receptor molecules (1 and 2) at sites I and II, respectively. Reduction and SDS-PAGE analysis does not lead to large changes in molecular mass of the complex. In (b) inefficient cross-linking at site II leads to a univalent hormone-receptor complex following analysis by reducing SDS-PAGE/autoradiography. In (c), proteolytic cleavage of the GH molecule followed by reduction of the inter-molecular disulphide bonds between the GH bps and the intra-molecular bonds in GH leads to the appearance of bp molecules bound univalently by GH fragments. Estimates of the molecular masses of the complexes formed are given. For the GHR, a value of 110 kDa (widely reported as the molecular mass of the GHR in various mouse fibroblast cell lines (35-37) has been assigned to the monomeric protein. Note that in (c) the GH molecule is depicted as proteolysed into two fragments of equal size. This is not intended to represent the situation in viva but is indicated as such here simply for clarity.

molecular mass forms most likely represent truncated forms of the GHR. Indeed there is evidence for the production of a soluble truncated form of the GHR suggested to arise (in a species-dependent manner) either by proteolysis of the full length GHR or alternate splicing of GHR mRNA [47,48]. Contamination of membrane preparations with this protein could lead to the appearance of low molecular mass bands. Alternatively, the presence of a previously uncharacterised short form of membrane-bound GHR (as has been described for the related prolactin receptor [49]) may be present. Indeed, very recent studies [50] have provided evidence for the presence of a short form of the GHR in particulate preparations from rat adipocytes. As a caveat to some of the arguments outlined above it is important to emphasise the importance of proteinase inhibitors in affinity cross-linking studies, especially if attempting to draw conclusions on the stoichiometry of hormone-receptor interaction based on apparent molecular mass of labelled bands following SDS-PAGE autoradiography. Although the majority of reported studies have included proteinase inhibitors in the preparation of membranes for cross-linking studies and also in the cross-linking reaction mix itself, this has not been universally the case and it is a consideration which should be borne in mind. Nonetheless, many of the hypotheses on the structure of the G H / G H R outlined above are testable and this is clearly an area which requires further examination. From the foregoing it is evident that the molecular heterogeneity of GH and GHR may lead to a corresponding variation in the nature of hormone receptor complexes. Thus, whilst the co-crystallisation and mechanistic studies of the Genentech group have provided invaluable information in the structural chemistry of the G H / G H R system, it remains possible that slight modifications in the chemistry of hormone-receptor interaction may occur. Analysis of these will require careful application of many techniques; affinity crosslinking, ligand blotting, high-performance gel filtration on receptors derived from many tissues and species using different naturally occurring variants of GH. Following this, questions may be asked as to how the biological significance of these different complexes may be addressed.

IV. Wider implications: the cytokine receptor superfamily Finally, as alluded to briefly above, the GHR belongs to a large and growing super-family of cytokine receptors. These include the GH and prolactin receptors (GHR and PRL-R), erythropoietin receptor (EPO-R) and receptors for interleukins (ILs) 3, 4, 6, 7 and 2/3 (the fl-subunit of the IL-2 receptor), granulocyte-monocyte colony-stimulating factor (GMCSF) and

mouse granulocyte colony-stimulating factor receptor (MG-CSF) [51,52]. In addition to this structural homology at the level of receptor, recently reported crystal structures for human IL-4 [53] and human GM-CSF [54] indicate that these hormones are folded as four a-helix bundles with a similar topography to GH. It is possible, therefore, that studies reported in this field may have relevance for the G H / G H R axis and vice versa. For example, by analogy with the GHR, soluble forms of various cytokine receptors have been reported [55]. In relation to this it is interesting to note that IL-3 appears to induce the phosphorylation and subsequent proteolysis of the /3-subunit of its receptor [56]. As it has been suggested that for some species the serum GHBP may be derived by proteolytic cleavage [47], it would be interesting to test the hypothesis that this is also ligand-induced in the G H / G H R axis. This may be important as it provides a mechanism other than receptor internalisation for down-regulation of receptor number and simultaneously provides a reserve of hormone in serum. Still in relation to IL-3, a recent study using mutants of the hormone reported two separate binding sites on IL-3 for receptor [57]. These proved to be located at the N- and C-terminus of the molecule and although distant in primary sequence were close together in the three-dimensional structure of the hormone. This is similar to the situation with GH (see Fig. 1A and B) and it may be that this model of asymmetric interaction of a polypeptide hormone with two receptor proteins is a common structural feature in this hormone-receptor family. Direct structural confirmation of this will come from X-ray crystallography of cytokine-receptor complexes. Although this is a substantial undertaking, the critical importance of cytokines as immunomodulators and the potential for pharmacological manipulation in this area may lead to such data being available in the near future. In relation to cytokine receptors, an important point to make is that although the EPO and G-CSF receptors are both homo-dimers (indeed the EPO receptor mutant R129C - where the native arginine residue at position 129 is replaced with cysteine, is constitutively (i.e., ligandindependently) active and is present as a disulphidelinked homodimer [58]) other cytokine receptors are heterodimers comprising a and /3 subunits with some evidence that the /3-subunit is common to different receptors (reviewed in Ref. 59). The evidence presented earlier in this review suggests that the G H / GHR belongs to the former category and that in a functional sense it might therefore be appropriate to sub-divide the cytokine family of receptors accordingly. In conclusion, extensive structural and functional data are now available for the GH/cytokine family of receptors and ligands. Analysis of these data and comparison across the family of receptor/ligands may lead to the discovery of some common signal-transduction

9 mechanisms. In this respect, study of receptors believed to require homodimerisation ,for activation may be most informative.

Acknowledgements The author thanks Maria Knight for preparation of this manuscript. This work was supported by the Scottish Office Agriculture and Fisheries Department.

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