Three characteristics of GH and GHR are worth noting from the evolutionary perspective. First, they provide a good system to study the coevolution of two proteins. As the functional pathway of GH begins with its binding to GHR, the two proteins are constrained to evolve together. Understanding the evolutionary trajectories of these two proteins will therefore help us understand how two proteins coevolve. Second, unlike nonprimate mammals, which possess only one GH gene, higher primates possess multiple copies of GH and GH-related genes, indicating the importance of GH gene duplication during the evolution of higher primates. Finally, GH shows a conspicuous pattern of "episodic" molecular evolution at the protein and DNA sequence level. For these reasons, GH and GHR provide an interesting case for the study of molecular evolution.
Another advantage of this system is that one can measure the effect of each amino acid substitution at the functional level (i.e., the effect on binding interactions between GH and GHR). Using in vitro binding assays, the contribution of each substitution on its phenotype (binding affinity) can be measured in a controlled environment. An example of this approach will be described.
Mammalian GH plays the role of a central endocrine regulator, controlling many different biochemical pathways (e.g., the metabolism of proteins, carbohydrates, and lipids). In humans, GH is also known to be involved in diabetes and to play a major role in carcinogenesis. Abnormal levels of GH directly induce specific phenotypes: dwarfism when hyposecreted and gigan-tism when hypersecreted, before puberty. In adults, hypersecretion of GH caused by pituitary adenomas leads to a condition, known as acromegaly, distinguished by large fingers, hands, and feet (see Okada and Kopchick, 2001 for a recent review).
The biochemical pathways induced by GH begin with the formation of a biologically active ternary complex comprised of one GH bound to two GHR molecules. First, one GH molecule binds to one GHR molecule through the high-affinity site of GH, called site1. Second, the resulting 1:1 complex then attracts a second GHR molecule to bind through the low-affinity site2 to form a 1:2 structure (Cunningham et al., 1989; Wells, 1996). This ternary complex is then able to elicit subsequent signal transduction pathways and participate in myriads of biochemical functions (Kossiakoff, 1995; Wells, 1994, 1996).
The study of GH and GHR interaction is greatly facilitated by a special characteristic of the GHR molecule. GHR is composed of three domains: extracellular, transmembrane, and intracellular. The part that participates in binding with the GH is the extracellular domain. This domain of GHR is found freely circulating in the bloodstream, independent of the other regions. When purified, they exhibit the same activity as the full-length counterpart (Fuh et al., 1990). Hence this domain is also called as the growth hormone binding protein (GHBP). Experiments to elucidate the structural and biochemical aspects of GH and GHR interactions can be performed using GHBPs, making experiments much more manageable.
The interfaces between molecules in the hGH-(hGHR)2 complex have been resolved in detail: structurally, by means of a high resolution X-ray crystallography (De Vos et al., 1992) and functionally, through mutational analyses (Cunningham and Wells, 1989; Cunningham et al., 1989; Clackson and Wells, 1995; Clackson et al., 1998). These two approaches generally agreed on the importance of specific residues. That is, the residues shown to reside structurally in the interfaces between the GH and GHR molecules had significant consequences in the stability of the GH-GHR complex when replaced with alanine—a relatively inert amino acid residue. This is because most of the residues in the binding interfaces form salt bridges and hydrogen bonds, which stabilize the intermolecular contact areas (De Vos et al., 1992; Kossiakoff, 1995). Recent studies of GH-GHR interactions suggest additional indirect contribution of some residues located relatively distant from the molecular interfaces (Behncken et al., 1997; Clackson et al., 1998).
Gene Duplications Leading to Multiple GH-Related Loci in Primates
Another characteristic of primate GH that may have significant evolutionary consequences is the presence of multiple GH-related genes. In human and rhesus monkey, there are five copies of GH and GH-related genes, while there is only a single GH-related gene in nonprimate mammals. In human, the five genes that comprise the GH cluster from 5' to 3' are: hGH-N, hPL-1, hPL-2, hGH-V, and hPL-3 (hPL stands for human placental lactogen: Figure 2). These genes show the same transcriptional orientation, but their expressions differ widely from one another in terms of both the tissues expressed and the level of transcription (Figure 2). Only hGH-N is produced in the pituitary and is referred as the GH, while all the other genes are expressed in the placenta. The locus hPL-1 carries a mutation in the 5' splice site so that only incompletely processed forms are produced and may not be functional (Barrera-Saldaña, 1998).
The questions of when and how the duplications occurred are parts of ongoing investigation. Adkins et al. (2001) showed that there is a single GH gene in the genome of the bushbaby—a prosimian. According to Wallis et al. (2001), there are multiple GH-related genes in the marmoset—a New World monkey. These studies indicate that the amplification of the GH gene cluster occurred after the separation of the haplorhine lineage from the strepsirhine lineage. However, it is unclear whether all the loci in human and rhesus monkey are the products of the same duplications as in the marmoset, or whether some duplications are unique to the human or rhesus monkey lineage. It is often difficult to infer the evolutionary relationships of duplicated genes because of the possibility of extensive gene conversion among loci (Wallis, 1996).
The fact that the GH gene has been duplicated to multiple copies in higher primates suggests selective advantage for the retention of duplicate GH genes.
hPL-2 hGH-V hPL-3
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