Mechanisms of P2Y 2 nucleotide receptor desensitization, implications for cystic fibrosis pharmacotherapy
P2Y receptors in normal and cystic fibrosis(CF) epithelia activate cMciumdependent chloride secretion in response to UTP. Receptor expression, regulation, and signal transduetion must be studied to reali,ze the full potential of the P2Y2 receptor as a target for UTP pharmacotherapy in overcoming defective chloride secretion in CF epithelia. Our studies show that the P2Y2 receptor desensitizes upon repeated exposure to UTP in CF epithelia. Similarly, a recombinant P2Y2 receptor expressed in human 1321NI astrocytoma cells desensitizes rapidly in response to 1 #M UTP, determined by measurements of intracellular calcium mobilization. Agonist-independent P2Y2 receptor desensitization occurred with phorbol ester treatment, which, with UTP-mediated desensitization was partially reduced with a specific protein kinase C(PKC) inhibitor. The desensitization of the P2Y2 receptor was investigated by mutagenesis of three consensus PKC sites and deletions of the carboxy-terminal tail. Deletion of a domain containing two PKC phosphorylation sites, and expression of the altered receptor in 1321N1 cells resulted in a 35% reduction of desensitization when compared to the wild type receptor. This altered receptor has a T1/2 of sequestration of 30-60 minutes compared to a TU2 of sequestration of 5-10 minutes for the wild type receptor. Other mutations of the P2Y2 receptor should delineate amino acids responsible for desensitization and sequestration, and provide information which may help to overcome this problem, so improving UTP therapies for CF. 2
Garrad, R. C., M. A. Otero, L. Erb, L. I. Clarke, F. Gonzalez, J. T. Turner, and G. A. Weisman. "Mechanisms of P2Y (2) nucleotide receptor desensitization, implications for cystic fibrosis pharmacotherapy." FASEB Journal11, no. 9 (1997): A1328-A1328.