The short and long forms of the alpha subunit of the stimulatory guanine-nucleotide-binding protein are unequally redistributed during (-)-isoproterenol-mediated desensitization of intact S49 lymphoma cells.
We report here that desensitization of the beta-adrenergic receptor-triggered transmembrane signalling in S49 wild-type lymphoma cells, induced by (-)-isoproterenol (1 microM), results in unequal intracellular redistribution of the splicing variants of the alpha subunit of the stimulatory guanine-nucleotide-binding regulatory (Gs alpha) protein (Gs alpha-short and Gs alpha-long) and alters the functional characteristics of the membrane-associated signal transduction complex. We found that two cellular pools of membranes, light-density membranes and plasma membranes prepared by sucrose-density-gradient centrifugation of cell homogenates differed in their content of Gs alpha splicing subforms and, moreover, that prolonged activation of the beta-adrenergic pathway induced intermembrane redistribution of the splicing variants of Gs alpha. Short (10 min) as well as prolonged (1 h) (-)-isoproterenol treatment of the cells shifted Gs alpha-short from light-density membranes to plasma membranes and increased the total amount of light-density membrane-bound Gs alpha-long; in parallel, the maximal (-)-isoproterenol-stimulated or AlF4(-)-stimulated adenylyl cyclase activities measured in the plasma membrane pools prepared from treated cells decreased. The functional characteristics of the membrane-bound Gs alpha pools were examined by a cyc(-)-reconstitutive adenylyl cyclase assay where extracts of the plasma membrane and light-density-membrane pools, respectively, were mixed with plasma membranes derived from the mutant S49 cell line, cyc-, lacking Gs alpha. The maximal cyc(-)-reconstitutive activities of the extracts prepared from light-density membranes of short-term as well as long-term desensitized cells increased compared to control cells. These findings may indicate differences in the functioning of the splicing variants of Gs alpha.
Kvapil
P
P
Wallenberg Laboratory for Cardiovascular Research, Gothenburg University, Sweden.
Novotny
J
J
Svoboda
P
P
Ransnäs
L A
LA
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology*; D008223:Lymphoma / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D014407:Tumor Cells, Cultured
D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol