Title:Co-evolution of the Opioid/Orphanin Gene Family and Cognate Receptor Gene Families. PI: Robert M. Dores. Institution : University of Denver
Communication among organ systems is accomplished by two intercellular communication systems, the nervous system and the endocrine system, that facilitates the flow of information throughout the body. In these communication networks, critical messages are carried by chemical signals (ligands) that interact in a highly selective manner with specific receptors on a target cell, a "lock and key" strategy. The origin of these ligand/receptor interactions are unknown, but the studies in this proposal will examine species from old lineages in order to reconstruct the transformations that have occurred in ligand-coding and receptor-coding genes during the evolution of distinct chemical communication networks. In order to study the co-evolution of ligand-coding genes and their corresponding receptor-coding genes this proposal will use the opioid/orphanin ligand-coding gene family (the source of opioid and melanocortin ligands), the opioid receptor-coding gene family and the melanocortin receptor gene family, which provide excellent models. The opioid/orphanin ligands influence analgesia, cardiovascular function, and some actions of the immune system, while the melanocortins are involved in chronic stress regulation, fat and glucose metabolism, and feeding behavior. Students will analyze species at critical branch points in the radiation of vertebrates such as: the lungfish, Neoceratodus forsteri, the white sturgeon, Acipenser transmontanus, the horn shark, Heterodontus francisci , the ratfish, Hydrolagus colliei, and the jawless vertebrates, Myxine glutinosa (hagfish) and Petromyzon marinus (lamprey). Studies will integrate the cloning of ligand coding genes with the cloning of their respective cognate receptor-coding genes in these species. The cloned receptors will be expressed in cell lines for binding study analysis, and the distribution of these receptors in the central nervous system and in peripheral tissues will be determined. These studies will reveal novel sequences for both ligands and receptors in the species that have been selected for these projects. Receptor sequence and ligand sequence databases will be created to identify amino acid motifs that could be modified in future site directed mutagenesis experiments to address structure/function related questions. These studies may lead to the development of analogs to the ligands (opioids and melanocortins) that may have therapeutic applications. Projects will be conducted by Ph.D. and MS graduate students and undergraduate honors students. Minority students will be recruited for summer REU (NSF Research Experience for Undergraduates) positions. Finally, a molecular cloning project related to this set of studies will be incorporated into the lab techniques course, BIOL 3655 "Molecular Neuroendocrinology" annually. This proposal fits into the area of Biological Systems Informatics in which questions of genome evolution can be addressed using genomic sequence database analyses. This proposal will both add information to a large ligand/receptor sequence database, as well as "data-mine" the database for new insights into receptor/ligand protein evolution and linked gene co-evolution.
The primary objective of my NSF-funded research program has been to understand the evolution of chemical communication networks in vertebrates. Interactions between the nervous system and the endocrine system result in neuroendocrine circuits that can respond to external and internal stimuli. Since 1988, my NSF-sponsored research program has focused on the evolution of chemical signals which belong to the opioid/orphanin gene family and the interactions of some of these chemical signals with their cognate receptors. To date this research has resulted in 77 peer reviewed articles that were co-authored with graduate students and undergraduates. Our primary focus has been on the pituitary hormones derived from one of the genes in the opioid/orphanin family, Proopiomelanocortin. These hormones are called "melanocortins" and include adrenocorticotropin (ACTH) and the melanocyte stimulating hormones (MSHs). The melanocortins bind to melanocortin receptors (MCRs) on the surface of target cells, and the result is the activation of those cells to perform several biological processes. Among the cartilaginous fishes, bony fishes and tetrapods (Figure 1), jawed vertebrate lineages that emerged approximately 450 million years ago, there are five subtypes of melanocortin receptors (MC1R, MC2R, MC3R, MC4R, MC5R). This grant has focused on understanding the evolution of the melanocortin-2 receptor (MC2R) and the interaction of this receptor with the melanocortins. MC2R is a component of the hypothalamus/pituitary/adrenal cortex/interrenal axis (HPA/I). In vertebrates the HPA/HPI axis integrates sensory input which results in the release of ACTH by the pituitary. ACTH binds to the MC2 receptor on either adrenal cortex cells (reptiles, birds, mammals) or interrenal cells (fish and amphibians) and these cells synthesis and release glucocorticoid steroids. Glucocorticoids are responsible for maintaining glucose and energy balance in cells in response to normal daily activities and chronic stress experiences. The interaction between ACTH and the MC2 receptor in bony fish and tetrapods presents both a puzzle and an enigma. The puzzle is that while the other melanocortin receptors (i.e., MC1R, MC3R, MC4R, MC5R) can be activated by either ACTH of the MSHs with varying degrees of efficacy, the MC2R of teleosts (modern bony fishes) and tetrapods can only be activated by ACTH. The enigma is that both teleost and tetrapod MC2 receptors require interaction with the accessory protein, MRAP, for functional activation to occur. In the absence of MRAP, MC2R will be degraded in the adrenal or interrenal cell. To address both questions we took a comparative approach. We began by analyzing the functional properties of an MC2 receptor in the genome of a cartilaginous fish, Callorhynchus milii, (elephant shark; Figure 2). We found that when the elephant shark MC2R was expressed in Chinese Hamster Ovary (CHO) cells in the absence of an endogenous MRAP, elephant shark MC2R could be visualized on the surface of the cell. We also discovered that the elephant shark MC2 receptor could be activated by either ACTH or the MSH-sized hormones. These two observations are summarized in the evolutionary scheme presented in Figure 3. Our operating hypothesis is that following the divergence of the ancestral cartilaginous fishes and the ancestral bony fished, mutations occurred in the ancestral MC2 gene which rendered this receptor MRAP dependent and redesigned the binding sites on the receptor to only recognize ACTH (Figure 4). The absence of a MC2R/MRAP interaction has a negative effect on fitness for humans, and mostly likely for teleosts and non-mammalian tetrapods as well. Comparative studies to determine which regions in ACTH are making contact with the MC2 receptor were conducted. Structure/function studies on a frog MC2R, and a rainbow trout MC2R are just being submitted for publication. These studies have led to the hypothesis that there are two binding site on MC2R. One binding site accommodates the HFRW motif in ACTH (Figure 4; blue region). This site has been previously identified in other melanocortin receptors. The new contribution from our studies is the proposed role of TM4, TM5 and extracellular loop 2 (EL2) in binding the KKRRP domain in ACTH. Site directed mutagenesis experiments were initiated at the close of funding for this grant. I am seeking funding from other sources to complete the characterization of this site. A purpose of basic research is to provide an information base for translational research. In this regard, we have submitted a provisional patent for an ACTH antagonist that may have application in clinical medicine and aquaculture. Training students is a major role for NSF grants, and the research of 8 graduate students and 10 undergraduates was supported by this grant. The procedures used in this research have also been incorporated into a lab technique course for undergraduates. These students can introduce hormone receptors into cells in culture and study the dynamic properties of these receptors in living cells.
