Agonist-induced desensitization of a P2Y-purinergic receptor-regulated phospholipase C.
A guanine nucleotide-dependent P2Y-purinergic receptor-regulated phospholipase C activity of turkey erythrocyte membranes has been characterized in detail previously (Boyer, J. L., Downes, C. P., and Harden, T. K. (1989) J. Biol. Chem. 264, 884-890). The occurrence of agonist-induced desensitization of this receptor-regulated phospholipase C is now described. Preincubation of turkey erythrocytes with the P2Y-purinergic receptor agonist ADP beta S resulted in a marked loss of capacity of ADP beta S plus GTP to stimulate phospholipase C in membranes derived from these cells. The half-time of occurrence of desensitization was 0.5-2.0 min, and within 10 min responsiveness had reached a new quasi-steady state level representing 40-55% of control. Transfer of agonist-preincubated erythrocytes to agonist-free medium resulted in recovery of agonist plus GTP responsiveness of the membrane phospholipase C activity to control levels with a half-time of 10-20 min. The change in ADP beta S plus GTP responsiveness occurred as a loss of maximal effect with little or no change in the apparent affinity of agonist for stimulation of inositol phosphate production. Induction of desensitization occurred with an agonist-specificity that followed that expected of a P2Y-purinergic receptor. Neither the rate of activation nor the final phospholipase C activity attained in the presence of GTP gamma S alone was altered in membranes from cells preincubated with ADP beta S for 15 min. AlF-4-stimulated inositol phosphate production was also not modified in membranes from agonist-preincubated erythrocytes. In contrast, the capacity of ADP beta S to increase the rate of activation of phospholipase C by GTP gamma S was markedly reduced in membranes from agonist-preincubated cells. The amount of 3H-radioactivity in phosphoinositides, as well as the ratio of labeling among the phosphoinositides, was not altered by incubation of erythrocytes with a P2Y-purinergic receptor agonist. Taken together these data suggest that P2Y-purinergic receptor agonist-induced desensitization occurs as a consequence of a modification at the level of the receptor or at the level of receptor-guanine nucleotide regulatory protein (G-protein) coupling with no change occurring in the capacity of the G-protein to activate phospholipase C.
Martin
M W
MW
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599.
Harden
T K
TK
D000244:Adenosine Diphosphate / Q000494:pharmacology / Q000031:analogs & derivatives; D000818:Animals; D004910:Erythrocyte Membrane / Q000201:enzymology / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000097:blood*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D010716:Phosphatidylinositols / Q000097:blood; D011983:Receptors, Purinergic / Q000187:drug effects / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology*; D014422:Turkeys; D010738:Type C Phospholipases / Q000097:blood*
D010716:Phosphatidylinositols; D011983:Receptors, Purinergic; D013873:Thionucleotides; C030812:adenosine 5'-O-(2-thiodiphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins