F96s

V144D R217X R161C R211G

Signal Extracellular Transmembrane Intracellular peptide domain domain domain

FIGURE 5-25 Structure of the GHR gene and mutations in Laron syndrome. The coding exons (boxes) are shown to approximate relative size; intron sizes are not to scale, and the noncoding part of exon 10 has been omitted. The structural protein domains encoded by the exons are indicated. The three shaded exons are involved in a deletion mutation. The locations (dots) and types of known point mutations within exons 4-7 are summarized above the diagram. The three splice site mutations are indicated by black bars at the exon 4/intron 4, intron 5/exon 6, and intron 6/exon 7 boundaries and are described below the diagram. Reproduced with permission from Harvey, S., and Hull, K. L. (1995). Growth Hormone Action: Growth Hormone Receptors. In "Growth Hormone" (S. Harvey, C. G. Scanes, and W. H. Daughaday, eds.), pp. 303-335. CRC Press, Boca Raton, FL.

Signal Extracellular Transmembrane Intracellular peptide domain domain domain

FIGURE 5-25 Structure of the GHR gene and mutations in Laron syndrome. The coding exons (boxes) are shown to approximate relative size; intron sizes are not to scale, and the noncoding part of exon 10 has been omitted. The structural protein domains encoded by the exons are indicated. The three shaded exons are involved in a deletion mutation. The locations (dots) and types of known point mutations within exons 4-7 are summarized above the diagram. The three splice site mutations are indicated by black bars at the exon 4/intron 4, intron 5/exon 6, and intron 6/exon 7 boundaries and are described below the diagram. Reproduced with permission from Harvey, S., and Hull, K. L. (1995). Growth Hormone Action: Growth Hormone Receptors. In "Growth Hormone" (S. Harvey, C. G. Scanes, and W. H. Daughaday, eds.), pp. 303-335. CRC Press, Boca Raton, FL.

described, to form the active homodimer through an identical site on the second receptor (GHrGHR2). Homodimer formation undoubtedly produces an altered conformation, so that the intracellular domains now associate with a tyrosine kinase of the Janus family (JAK2). The intracellular receptor domains probably become phosphorylated by JAK2 and other substrates also become phosphorylated, such as MAP kinase and others. The end result is gene activators in the nucleus that may involve a number of transactivating and other factors, such as Spi 2.1, c-/os, and c-jun, affecting transcriptional activation of genes for IGF-1, P450SC, MUP (major urinary proteins), and others, probably including IGF-binding proteins. The activated transcription of IGF-I is key to the signal transduction mechanism initiated by GH for the growth of tissues. At best our knowledge of this signal transduction mechanism is sketchy. There is also information suggesting that, following GHR homodimer formation, phospholipase C may become activated to produce diacylglycerol and IP3, which would lead to protein kinase C activation and phosphorylations affecting transcription factors acting in the nucleus. The phosphorylation of GHR in the process of cytoplasmic interaction with JAK2 is different, so far, with our current knowledge of the fate of the PRL receptor homodimer for which there is no strong evidence for its phosphorylation. This series of events leads to the production of IGF-I by the liver and other tissues, which is a key effector (somatomedins) of tissue growth.

E. Growth Hormone and Somatomedins

The relationship between GH and somatomedins (IGFs) is demonstrated in Figure 5-27. Three somatomedins are formed in the human, A, B, and C. One of these (somatomedin C) is identical to IGF-I. Once secreted into the bloodstream, GH binds to a specific receptor on the hepatocyte cell membrane and triggers a second message. In response, the hepatocyte (see Chapter 10 for cell biology of the hepatocyte) either synthesizes or releases preformed somatomedins into the blood, and the hepatocyte also produces a soma-tomedin-binding protein of ~50,000 molecular weight. IGF-binding proteins are summarized in Chapter 19 (Growth Factors). The somatomedins are —7000 molecular weight and circulate mostly complexed to a binding protein. There are specific somatomedin receptors on liver cells and cells of adipose, lymphocytes, bone, placental membrane, and others, and these receptors are distinct from insulin receptors as revealed by competition experiments. The interaction of free somatomedin (not the somatomedin-binding protein complex) with its receptor triggers a second messenger, resulting in a mitogenic effect. The second messenger may occur through stimulation of a tyrosine kinase, which is part of the IGF receptor that probably phos-phorylates IRS-1 (the insulin receptor substrate) and perhaps other proteins. The sequences of IGF-I, IGF-II, and insulin are shown in Figure 5-28. Because of the rather extensive homology of somatomedins with

FIGURE 5-26 Probable location of two sites reactive with the GHR projected on a model of the GH structure. The cyclinders represent four predominantly helical domains. Alanine substitutions in hGH that disrupt binding of hGHbp at either site 1 or site 2 are generally delineated by the large shaded circles. Residues for which alanine mutants reduce site 2 binding by 2-4-fold, 4-10-fold, 10-50-fold, and more than 50-fold are shown by increasingly larger squares (■, and I, respectively). Residues marked by the symbols •, •, •, and O

represent sites where Ala mutations in site 1 of hGH cause reductions of 2-4-fold, 4-10-fold, greater than 10-fold, or a 4-fold increase in binding affinity for the hGHbp, respectively, using the MAb 5 immunoprecipitation assay. Reprinted from Cunningham, B. C., Ultsch, M., de Vos, A. M., et al. (1991). Dimerization of the extracellular domain of the human growth hormone receptor: Crystal structure of the complex. Science 254,821-825, with permission of the American Association for the Advancement of Science). Reprinted with permission from Daughaday, W. H. (1995). Growth hormone, insulin-like growth factors, and acromegaly. In "Endocrinology" (L. J. De Groot, ed.), 3rd ed„ Vol. 1, pp. 303-329. W. B. Saunders Co., Philadelphia, PA.

FIGURE 5-26 Probable location of two sites reactive with the GHR projected on a model of the GH structure. The cyclinders represent four predominantly helical domains. Alanine substitutions in hGH that disrupt binding of hGHbp at either site 1 or site 2 are generally delineated by the large shaded circles. Residues for which alanine mutants reduce site 2 binding by 2-4-fold, 4-10-fold, 10-50-fold, and more than 50-fold are shown by increasingly larger squares (■, and I, respectively). Residues marked by the symbols •, •, •, and O

represent sites where Ala mutations in site 1 of hGH cause reductions of 2-4-fold, 4-10-fold, greater than 10-fold, or a 4-fold increase in binding affinity for the hGHbp, respectively, using the MAb 5 immunoprecipitation assay. Reprinted from Cunningham, B. C., Ultsch, M., de Vos, A. M., et al. (1991). Dimerization of the extracellular domain of the human growth hormone receptor: Crystal structure of the complex. Science 254,821-825, with permission of the American Association for the Advancement of Science). Reprinted with permission from Daughaday, W. H. (1995). Growth hormone, insulin-like growth factors, and acromegaly. In "Endocrinology" (L. J. De Groot, ed.), 3rd ed„ Vol. 1, pp. 303-329. W. B. Saunders Co., Philadelphia, PA.

insulin, it has been deduced that both hormones derive from the same ancestral gene. The growth-promoting activity of insulin itself is due to its limited ability to bind to IGF-I receptor. To do this, insulin has to be present in high concentrations because the affinity of the IGF-I receptor for insulin is about 1000 X less than the affinity of the insulin receptor for insulin. IGF-II can also productively bind to the IGF-I receptor, albeit with much lower affinity. Thus, all three factors are growth-promoting, and IGF-II and insulin become important when IGF-I is low or absent. Presumably, IGFs can cross over to the insulin receptor in the absence of insulin, although this sort of rescue may not be important in insulin-deficient diabetes.

Interestingly, as shown in Figure 5-29, the level of circulating somatomedin increases to a maximum until about 8 years of age when the human adult level is reached. Since growth continues for many years be yond age 8, it is obvious that the amount of circulating somatomedins is not rate-limiting for growth. Thus, some other component of the overall system may limit growth, such as the development of somatomedin receptors or some other factor involved in the utilization of these mitogens. Somatomedins also stimulate sulfation, and this is important to bone growth.

F. Growth Hormone Signal Transduction

The 130-kDa growth hormone receptor contains a single transmembrane domain with a glycosylated extracellular domain (Figure 5-30). While the cytoplasmic domain becomes phosphorylated on tryr osine residues, this domain is different from other growth factor receptors, like those for EGF and PDGF, and has a different tyrosine kinase domain. Interestingly, the human GH receptor is specific for human GH and does not bind GH

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