The long term goal of our research is to understand the mechanism of signal transduction by neuropeptide receptors. Substance-K (neurokinin-A) receptors play important roles in both normal and pathological processes, including memory retention and rheumatoid arthritis [l, 2, 3]. The goal of the research proposed here is to determine structure-function relationships of neuropeptide receptors using Neurokinin A receptor as a model system for neuropeptide receptors, which have seven hydrophobic membrane spanning regions. Following ligand binding, neuropeptide receptors couple with G-proteins to transduce their signals. A key breakthrough in analyzing structure-function is the cloning of the receptor. The human Neurokinin A receptor has been cloned by us and will be used in this analysis.
Specific aims are 1) To determine functional domains of the substance-K receptor involved with G-protein interaction by producing chimeras between the neurokinin A receptor and the beta2- adrenergic receptor which utilize different G-proteins. Precedence from the adrenergic and muscarinic receptor systems will guide the choice of domains to be swapped. 2) To determine the role of post-translational modification of the Neurokinin A receptor. a) There are potential myristylation and palmitoylation sites on the Neurokinin A receptor. The use and importance of these sites will be analyzed by metabolic labelling experiments and site directed mutagenesis. b) The response to neurokinin A is rapidly desensitized. In the adrenergic system, desensitization has been shown to controlled by phosphorylation of the C-terminal tail. The role of phosphorylation of the C-terminal tail of the Neurokinin A receptor will be analyzed by in vivo labelling with 32p and by deletion mutagenesis of the C-terminal tail. A concise understanding of the sites important in the interaction between neuropeptide receptors and their transducing proteins (G-proteins) is necessary for understanding the mechanism of signal transduction by neuropeptide receptors. The site of G-protein interaction and the role of post-translational modifications of the Neurokinin A receptor are keys in understanding the mechanism of stimulation or desensitization of the response to Neurokinin A, important concepts in normal regulation of the proper receptor function. The understanding of mechanism of signal transduction by these receptors may lead to more potent drugs for treating diseases such as rheumatoid arthritis and for analysis of memory retention induced b Neurokinin A.
| Cyr, C R; Devi, L A; Rudy, B et al. (1996) Heterologous desensitization of the human endothelin A and neurokinin A receptors in Xenopus laevis oocytes. Recept Signal Transduct 6:99-109 |
| Josiah, S M; Cyr, C R; Chu, V et al. (1994) Regions of the human neurokinin A receptor involved in the generation of second messengers and in receptor desensitization. Biochem Biophys Res Commun 199:626-32 |
| Cyr, C R; Rudy, B; Kris, R M (1993) Prolonged desensitization of the human endothelin A receptor in Xenopus oocytes. Comparative studies with the human neurokinin A receptor. J Biol Chem 268:26071-4 |
