In Vivo Analysis of Growth Hormone Receptor Signaling Domains and Their Associated Transcripts

Abstract

The growth hormone receptor (GHR) is a critical regulator of postnatal growth and metabolism. However, the GHR signaling domains and pathways that regulate these processes in vivo are not defined. We report the first knock-in mouse models with deletions of specific domains of the receptor that are required for its in vivo actions. Mice expressing truncations at residue m569 (plus Y539/545-F) and at residue m391 displayed a progressive impairment of postnatal growth with receptor truncation. Moreover, after 4 months of age, marked male obesity was observed in both mutant 569 and mutant 391 and was associated with hyperglycemia. Both mutants activated hepatic JAK2 and ERK2, whereas STAT5 phosphorylation was substantially decreased for mutant 569 and absent from mutant 391, correlating with loss of IGF-1 expression and reduction in growth. Microarray analysis of these and GHR؊/؊ mice demonstrated that particular signaling domains are responsible for the regulation of different target genes and revealed novel actions of growth hormone. These mice represent the first step in delineating the domains of the GHR regulating body growth and composition and the transcripts associated with these domains.Over the last decade, extensive in vitro studies have identified signaling pathways triggered by growth hormone receptor (GHR) activation and the sequence motifs within the conserved cytoplasmic domain of the receptor that are required to initiate these pathways. These studies have established the critical signaling role of JAK2 tyrosine kinase, which is recruited to the trimeric GHR2:GH complex at the box 1/2 motif (9). Hormone binding initiates JAK2 transphosphorylation and activation, resulting in phosphorylation of critical tyrosines within the cytoplasmic domain of the GHR, as well as other direct JAK2 substrates such as IRS-1 and -2. The distal cytoplasmic phosphotyrosines of GHR have been shown to recruit signal transducer and activator of transcription 5 (STAT5) and other proteins through SH2-domain interactions, whereas the proximal JAK2 activation domain is responsible for ERK1/2 and phosphatidylinositol 3-kinase (PI 3-kinase) activation (30), although it has been claimed that residues distal to m390 are required for ERK1/2 activation (residue 390, mouse sequence numbers given throughout) (38). There is some dispute regarding the distal tyrosine residues used to recruit STAT5 for activation, notably in relation to tyrosine m498 (8, 33); moreover, it has been claimed that tyrosines m341 and m346, proximal to boxes 1 and 2, may also generate active STAT5 (31).In vitro studies have identified other signaling elements within the distal region of the GHR cytoplasmic domain, for example, a JAK2-independent calcium signaling element between residues m465 and m517 (30). SHP2 phosphatase can have a dual role when bound to the cytoplasmic domain of the GHR. It binds primarily to m606 to attenuate JAK2-STAT5 signaling but can also serve as an adaptor protein (30). GHdriven activation of STAT5 can also be attenuated by suppres...

New Insights into Growth Hormone Receptor Function and Clinical Implications

“…Moreover the gene expression profile in these animals has similarities with that of aging rats, implying that the GH deficiency in aging is similar to GH deficiency. The linking of different signaling pathways to metabolic responses was possible after the generation of the GHR KI mice [12]. In this study the mutations in the ICD of the GHR facilitated the identification of target genes for particular signaling pathways and their effects on metabolism in the long term.…”

“…Double STAT5a/b–/– mice are further decreased in size in both sexes [21]. Mutations in the ICD of the GHR which prevent STAT5 activation decrease postnatal growth of both sexes by up to 50% in mutant 391, comparable to the 55% decrease in GHR–/– mice [12] (2, 3, 4). This decrease in postnatal growth occurs because STAT5b regulates expression of Igf-1 , as evident in recent promoter analysis studies [22] as well as Igfbp3 and Igfals [12], which stabilize IGF-1 in circulation.…”

“…Studies by Miller et al [18] and Rowland et al [12] identified dysregulated genes because of a lack of GH signaling. Moreover the gene expression profile in these animals has similarities with that of aging rats, implying that the GH deficiency in aging is similar to GH deficiency.…”

“…Transcripts for metabolic enzymes, particularly involving fat metabolism and energy metabolism, are significantly altered by GH in the liver [12,14,15,16] (table 1). The general features of all these studies are low fold changes (majority up to 2.5-fold), with steroid and drug-metabolizing enzymes being the most changed (above 2.5-fold).…”

“…In vitro, while numerous studies have mapped the cytoplasmic domains of the GHR used for controlling signaling pathways [2], only a few GH-regulated genes have been subjected to promoter analyses to define the signaling pathways responsible for regulation of their expression by GH. This has changed recently as a result of the application of microarray technology to identify GH target genes combined with genetically engineered rodent models [12]. As stature and metabolism can really be only studied in vivo, these studies used animal models to determine which genes are being regulated by GH.…”