Our goals are to use the mammalian growth hormone (GH) and its receptor (GHR) as a model system to study the process and mechanisms of molecular coevolution and to understand the molecular basis of GH-GHR interaction. Mammalian GHs and GHRs exhibit large diversity and species specificity. For example, human GH and GHR have peculiar features: (1) Human GH differ greatly in sequence from nonprimate GHs. (2) Human GH stimulates growth in nonprimates, whereas nonprimate GHs are inactive in humans (known as the species specificity of human GHR). (3) Nonprimate mammals have only one GH gene, whereas humans have 5 GH-like genes. Peculiar features of GH and GHR are also found in other mammals such as rat, mouse, and cow. In particular, guinea pig GHR does not bind to human (or bovine) GH, and guinea pigs grow well even in the absence of GH. To understand these and other features, we need to study the molecular basis of the GH-GHR interaction. We propose to pursue the following work, using a combination of molecular, biochemical, evolutionary, and statistical approaches: 1. Determine the numbers of GH and GH-like genes in the galago, tarsier, squirrel monkey, and guinea pig. Sequence the GH and GH-like genes in these species and the GH gene in several primates and the guinea pig. 2. Sequence the GHR gene in the galago, tarsier, squirrel monkey (and, partially, other higher primates), the rabbit, and guinea pig. 3. Express GH and GH binding protein (GHBP) cDNAs from various species. 4. Identify sites in GH and GHR that are potentially functionally important and perform specific mutagenesis to generate mutants. 5. Use the variant GHs and GHBPs in competitive binding assays to determine the effect of each change. For amino acid differences with observed significant effects on binding, determine their collective effect in combinations. 6. Conduct statistical analyses of data to characterize evolution of GH and GHR.
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