The D152H mutation found in growth hormone insensitivity syndrome impairs expression and function of human growth hormone receptor but is silent in rat receptor

Abstract

In two patients with growth hormone (GH) insensitivity syndrome (Laron syndrome), in whom the GH receptor is able to bind the hormone, the D152H mutation was identified, and lack of dimerization was proposed to explain GH resistance in these patients. To examine further the consequences of the substitution of conserved aspartate 152 on the function of the GH receptor (GHR), we reproduced the mutation in vitro on the full length GH receptor cDNA from man and rat. Effects of the mutation on expression and activity of the GHR were analyzed in 293 cells transfected with wild-type and mutant GHR cDNAs. Mutant human receptor protein was expressed at a lower level than wild-type receptor and its activity was reduced: GH-dependent signal transducer and activator of transcription 5 (Stat5)-mediated transactivation of a reporter gene was lower in 293 cells transfected with mutant GHR cDNA than in transfected cells expressing a comparable level of wild-type GHR. The membrane-bound form of the mutant and of the wild-type human GHR were able to homodimerize, as suggested by the size of the complexes detected in cross-linking experiments with 125 I-human (h) GH, and also by the activity in the functional test. With the soluble GHR resulting from proteolysis of the wild-type membrane form, no dimeric complexes could be detected. However, when a soluble receptor lacking the transmembrane and cytoplasmic domains of the receptor was expressed, wild-type and not mutant GH binding protein (GHBP) was able to form dimers in the presence of hGH. The amino acid substitution has no effect on either expression or function of the rat receptor. Structural modeling of D152H soluble human and rat GHR (GHBP) supports the species-specific functional consequences of the mutation. Evaluation of the functional importance of the mutation strongly suggests that impairment in expression and activity of the mutant receptor, rather than complete lack of dimerization, explains the GH resistance of the patients.

Dimerization and Signal Transduction of the Growth Hormone Receptor

“…We assumed that the mutations cause local structural perturbations, as was proposed for the D152H mutant (29). Because crystallographic data of these mutant GHRs are not available, we applied our protease-protection assay to investigate the effect of the mutations on the integrity of the GH-(GHR) 2 complex (Fig.…”

“…Previously, species specificity has been observed for the D152H mutation, which prevents signal transduction via the human GHR, but it is silent in rat (29). Therefore, we examined the tyrosine phosphorylation of the GHR mutants in our particular cell system.…”

“…However, GHR(D152H) is able to bind GH and contains an intact JAK2 binding region (26). Initially, the D152H mutation was proposed to affect receptor dimerization, but cross-linking of radiolabeled GH to GHR(D152H) revealed a GH-(GHR) 2 complex (29). Rather than affecting dimerization, the D152H mutation was suggested to alter the conformation of the GHR.…”

Structurally distinct membrane-associated and soluble forms of GH-binding protein in the mouse

“…The mutant human GHR is characterized by defects in expression and activity. The same mutation introduced into rat GHR has no effect on expression or activity of rat GHR (Esposito et al 1998). One possible interpretation of this finding is that in the rat (and the mouse), this highly conserved residue is dispensable for GHR but required for proper function of GHBP.…”

“…One of the five potential sites for the addition of N-glycans to mouse GHBP (asparagine 145) is also exposed on the surface in close proximity to the RGD sequence. A protein bound to the RGD sequence would not directly affect the GH-binding site of GHBP, but would interfere with GH-induced dimerization of GHBP since the corresponding residues of human GHBP (HAD) and rat GHBP (RGD) are in the contact region between the two GHBPs in the 1:2 GH:GHBP complex (Esposito et al 1998).…”