Expression of phosducin in a phosducin-negative cell line reveals functions of a Gbetagamma-binding protein.
Phosducin is a member of the large group of proteins that bind to G-protein betagamma-subunits (Gbetagamma) and whose biological functions are often unknown. Human A431 cells do not contain detectable amounts of phosducin. We generated A431 cells expressing phosducin at a level of approximately 1 pmol/mg of cytosolic protein, which is approximately 10% of the phosducin level in brain. cAMP accumulation in response to beta2-adrenergic receptor agonists was enhanced at early times in phosducin-expressing cells, but reached a lower plateau than in control cells. Permeabilization of the cells with digitonin did not change this pattern, but allowed the introduction of specific inhibitors: antibodies to phosducin abolished all differences between the two cell lines. Inhibitors of the beta-adrenergic receptor kinase abolished the differences at early time points. An almost complete loss of beta2-adrenergic receptor desensitization in the phosducin-expressing cells was also observed when intact cells were desensitized and receptor function was then determined in membrane preparations. Inhibition of protein kinase A accentuated the effects of phosducin, suggesting that also in vivo phosducin is regulated by this kinase. These data indicate that phosducin affects G-protein-mediated signaling in at least two ways: it dampens the overall responsiveness, and it impairs the rapid desensitization mediated by the beta-adrenergic receptor kinase.
Schulz
K
K
Institute for Pharmacology, University of Würzburg, Versbacher Strabetae 9, D-97078 Würzburg, Federal Republic of Germany.
Danner
S
S
Bauer
P
P
Schröder
S
S
Lohse
M J
MJ
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D005136:Eye Proteins / Q000096:biosynthesis*; D020555:GTP-Binding Protein Regulators; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008969:Molecular Sequence Data; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010750:Phosphoproteins / Q000096:biosynthesis*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D000318:Adrenergic beta-Agonists; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D010726:Phosphodiesterase Inhibitors; D010750:Phosphoproteins; D011943:Receptors, Adrenergic, beta; C065631:phosducin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D015056:1-Methyl-3-isobutylxanthine