| Abstract
|
:
|
P2Y receptors are G protein-coupled receptors activated by extracellular nucleotides. Although eleven receptors have been proposed to be P2Y receptors, only five usd\rm (P2Y\sb1,\ P2Y\sb2,\ P2Y\sb4,\ P2Y\sb6,usd and P2Y) have been shown to be functional mammalian P2Y receptors. This dissertation presents the molecular cloning and pharmacological characterization of the turkey and human homologues of the P2Y receptor, analysis of two orphan G protein-coupled receptors (the 6H1/p2y5 and U2 receptors) that had low but significant homology to known P2Y receptors, and an in depth characterization and search for a human homologue of the avian p2y3 receptor. The turkey and human P2Y receptor homologues were cloned, stably expressed, and characterized for their nucleotide selectivities and second messenger signaling properties. The turkey P2Y receptor was shown to be the same receptor that is expressed in turkey erythrocytes by virtue of their nearly identical nucleotide selectivities and by the demonstration of the abundant expression of P2Y receptor mRNA in turkey erythrocytes. Furthermore, the human P2Y receptor was shown to have a nucleotide selectivity very similar to that of the turkey P2Y receptor. In the second part of my dissertation, the possibility that two orphan G protein-coupled receptors, the 6H1/p2y5 receptor and the U2 receptor, were P2Y receptors was investigated by stably expressing epitope-tagged receptors in 1321N1 cells, verifying their cell-surface expression, and determining their nucleotide-promoted second messenger responses. No second messenger responses in cells expressing either receptor could be observed to a variety of nucleotides under conditions in which their cell surface expression was verified by an intact cell based ELISA. Thus, neither of these receptors are members of the P2Y family of signaling proteins. In the last part of my dissertation, the relationship of the avian p2y3 receptor to mammalian P2Y6 receptors was investigated. Pharmacological studies of the two receptors expressed in the same cell line indicated that both of these receptors are UDP-selective receptors with similar but not identical nucleotide selectivities. In addition, no gene in the human genome could be identified that was more homologous to the avian p2y3 than the human P2Y6 receptor. These data indicate that the avian p2y3 receptor is a species homologue of the mammalian P2Y6 receptor.
|