Growth Hormone Receptor: In Vivo Analysis of the Cytoplasmic Signaling Domains
© 2005 Prime Health Consultants, Inc.
In vitro studies of the growth hormone receptor (GHR) have identified multiple post-receptor signaling pathways including JAK2 tyrosine kinase, STAT5, ERK1/2, PI3-kinase, a JAK2-independent calcium signaling element, SHP2 phosphatase, SOCS and CIS. Although STAT5 is primarily responsible for GH-induced expression of insulin-like growth factor (IGF)-I, STAT5b-/- mice have less severe growth retardation than GHR-/- mice, indicating a physiologic significance of alternative pathways.
Rowland and colleagues undertook the impressive task of teasing apart the GHR signaling domains in vivo. They created 2 knockin mice bearing truncated GHR mutants: m569 was truncated at residue 569 (wild-type GHR contains 650 amino acids) and had site-directed mutations of tyrosines 539 and 545 in order to delete 70% of the STAT5 docking sites, while m391 was truncated at residue 391, thereby also deleting the proximal STAT5 sites (0% STAT5 signaling left) while retaining 100% of JAK2 and ERK1/2 signaling. Radioreceptor assays confirmed normal levels of specific binding of 125I-labeled GH, expressed per milligram of membrane protein, in the 2 mutant mice. Comparison to wild-type mice showed 44% of GH-dependent growth in the m569 mice and 11% growth in the m391 mice. Serum IGF-I levels were 16% to 21% of wild-type in m569 and less than 10% in m391. However, hepatic IGF-I transcript levels were not depressed as much, suggesting additional IGF-I protein clearance due to decreased ternary complex formation from reductions in IGFBP-3 and ALS expression. Both mutants developed obesity in males after 4 months of age, as well as associated hyperglycemia.
The authors took their characterizations one step further: microarray analysis of the 2 mutant mice compared to wild-type and GHR-/- mice revealed domain-specific regulation of different target genes. Four hundred three transcripts (398 genes) were differentially expressed across all groups, 20 were common to all, 13 unique to m569, 59 unique to m391 and 268 unique to GHR-/-. Interestingly, only 5 genes were regulated exclusively by residues 569-650; thus the distal 70% STAT5 binding played a minor role in mediating the genomic effects of GH. IGF-I was one of 20 STAT5-regulated genes, and the proximal 30% STAT5 binding was important for inducing IGF-I. The majority of regulated transcripts related to the more proximal GHR domains, where JAK2 leads to ERK1/2 and PI3-kinase signaling and SOCS proteins play an inhibitory role. These included many metabolic genes and genes related to hepatocyte function such as signaling, proliferation, translation, and transporter proteins. I refer the reader to the paper and the associated website (http://research.imb.uq.edu.au/~mwaters/ghr/) for detailed listings.
Editor’s Comment: This tremendous piece of work significantly advances our knowledge of GHR function; not only is GHR signaling dissected to a sharper degree than before, but new GH functions are suggested by the target genes identified in the microarray analyses. How does all this correlate clinically? GHR mutations cause GH-insensitivity syndrome, or Laron syndrome, characterized by severe postnatal growth retardation, low circulating IGF-I levels despite elevated GH levels, and lack of IGF-I response to rhGH. The majority of reported mutations occur in the extracellular part of the protein; defects in the cytoplasmic domains of GHR, studied in this paper, are rare. However, 2 recent papers described patients with distal cytoplasmic GHR mutations resulting in selective loss of STAT5 pathway. Two siblings, in their 50’s, had homozygous deletions that encoded GHRs truncated at amino acid 449; loss of STAT5 binding, despite retention of normal JAK2 phosphorylation, STAT3 and ERK2, was sufficient to cause severe growth failure (height z scores of –8.7 and –6).1 A 17-year-old girl was identified with a height z score of –5.28 and classic features of Laron syndrome. She was a compound heterozygote for novel GHR mutations: C83X (lack of GHR expression due to mRNA decay or defect in cell membrane anchoring) and 1776del (GHR truncated at 581 amino acids). The 1776del GHR had significant impairment of STAT5 activation despite intact extracellular, transmembrane and more than 80% of the cytoplasmic GHR domains. STAT3 activation was normal.2 The clinical importance of STAT5 signaling was further confirmed in a 16.5-year-old girl whose Laron syndrome was caused not by a GHR mutation, but by a homozygous missense mutation in the STAT5b gene; her height z score3 was –7.5.
Adda Grimberg, MD
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