Cell-free heterologous desensitization of adenylyl cyclase in S49 lymphoma cell membranes mediated by cAMP-dependent protein kinase.
We have examined the cell-free heterologous desensitization of adenylyl cyclase in plasma membrane preparations from S49 wild-type (WT) and kin- cells (which lack cAMP-dependent protein kinase) incubated with purified catalytic subunit of cAMP-dependent protein kinase (cA.PKc). cA.PKc caused a rapid (t1/2 = 40 s) decrease in the hormone responsiveness of adenylyl cyclase in the WT membrane preparations that mimicked the intact cell heterologous desensitization; that is, there was an increase in the Kact for both epinephrine and prostaglandin E1 (PGE1) stimulations of adenylyl cyclase induced at the receptor level because neither forskolin- nor NaF-stimulated activity was affected. The desensitization was independent of agonist occupancy of the receptor, and the effects were blocked both by the active fragment (amino acids 5-22) of the specific inhibitor of cA.PK and by p[NH]ppA. cA.PKc treatment of kin- membranes resulted in a heterologous desensitization that resembled the effects of WT adenylyl cyclase, with the exception that forskolin-stimulated activity was also reproducibly decreased by 24%. cA.PKc had no effect on WT membranes isolated from cells that had previously undergone maximal heterologous desensitization during treatment with 10 microM forskolin. In contrast, cA.PKc-induced heterologous desensitization of kin- membranes was additive with the epinephrine-induced homologous desensitization of intact cells. Cell-free desensitizations were reversed by incubation of membranes with cA.PKc and ADP, conditions that drive the kinase reaction backward. The similarities of our cell-free cA.PKc-mediated heterologous desensitization of adenylyl cyclase with the intact cell desensitization support our hypothesis that heterologous desensitization of the WT lymphoma cells is mediated by cA.PK via a mechanism independent of homologous desensitization.
Kunkel
M W
MW
Graduate School of Biomedical Sciences, University of Texas Health Science Center, Houston 77225.
Friedman
J
J
Shenolikar
S
S
Clark
R B
RB
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000527:Alprostadil / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D002474:Cell-Free System; D005576:Colforsin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004837:Epinephrine / Q000494:pharmacology; D066298:In Vitro Techniques; D008223:Lymphoma; D011494:Protein Kinases / Q000502:physiology*; D011943:Receptors, Adrenergic, beta / Q000502:physiology; D014407:Tumor Cells, Cultured
D011943:Receptors, Adrenergic, beta; D005576:Colforsin; D000244:Adenosine Diphosphate; D011494:Protein Kinases; D000262:Adenylyl Cyclases; D000527:Alprostadil; D004837:Epinephrine