Phosphorylation of the G protein gamma12 subunit regulates effector specificity.
Although the G protein betagamma dimer is an important mediator in cell signaling, the mechanisms regulating its activity have not been widely investigated. The gamma12 subunit is a known substrate for protein kinase C, suggesting phosphorylation as a potential regulatory mechanism. Therefore, recombinant beta1 gamma12 dimers were overexpressed using the baculovirus/Sf9 insect cell system, purified, and phosphorylated stoichiometrically with protein kinase C alpha. Their ability to support coupling of the Gi1 alpha subunit to the A1 adenosine receptor and to activate type II adenylyl cyclase or phospholipase C-beta was examined. Phosphorylation of the beta1 gamma12 dimer increased its potency in the receptor coupling assay from 6.4 to 1 nM, changed the Kact for stimulation of type II adenylyl cyclase from 14 to 37 nM, and decreased its maximal efficacy by 50%. In contrast, phosphorylation of the dimer had no effect on its ability to activate phospholipase C-beta. The native beta1gamma10 dimer, which has 4 similar amino acids in the phosphorylation site at the N terminus, was not phosphorylated by protein kinase C alpha. Creation of a phosphorylation site in the N terminus of the protein (Gly4 --> Lys) resulted in a beta1 gamma10G4K dimer which could be phosphorylated. The activities of this beta gamma dimer were similar to those of the phosphorylated beta1 gamma12 dimer. Thus, phosphorylation of the beta1 gamma12 dimer on the gamma subunit with protein kinase C alpha regulates its activity in an effector-specific fashion. Because the gamma12 subunit is widely expressed, phosphorylation may be an important mechanism for integration of the multiple signals generated by receptor activation.
Yasuda
H
H
Department of Pharmacology, Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908, USA.
Lindorfer
M A
MA
Myung
C S
CS
Garrison
J C
JC
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D054799:Phospholipase C beta; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000378:metabolism
D007527:Isoenzymes; D000242:Cyclic AMP; D011493:Protein Kinase C; D010738:Type C Phospholipases; C518033:PLCB1 protein, human; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases