Loss of association between activated Galpha q and Gbetagamma disrupts receptor-dependent and receptor-independent signaling.
The G protein subunit, betagamma, plays an important role in targeting alpha subunits to the plasma membrane and is essential for binding and activation of the heterotrimer by heptahelical receptors. Mutation of residues in the N-terminal alpha-helix of alpha s and alpha q that contact betagamma in the crystal structure of alpha i reduces binding between alpha and betagamma, inhibits plasma membrane targeting and palmitoylation of the alpha subunit, and results in G proteins that fail to couple receptor activation to stimulation of effector. Overexpression of betagamma can recover this loss of signaling through Gs but not Gq. In fact, a single mutation (I25A) in alpha q can block alpha q-mediated generation of inositol phosphates. Function is not recovered by betagamma overexpression nor myristoylation directed plasma membrane localization. Introduction of a Q209L activating mutation with I25A results in a constitutively active alpha q as expected, but surprisingly a R183C activating mutation does not result in constitutive activity when present with I25A. Examination of binding between alpha and betagamma via a pull down assay shows that the N-terminal betagamma-binding mutations inhibit alpha-betagamma binding significantly more than the R183C or Q209L activating mutations do. Moreover, introduction of the I25A mutation into alpha q RC disrupts co-immunoprecipitation with PLCbeta1. Taken together, results presented here suggest that alpha-betagamma binding is necessary at a point downstream from receptor activation of the heterotrimeric G protein for signal transduction by alpha q.
Evanko
Daniel S
DS
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, 233 S. 10th St., 839 BLSB, Philadelphia, PA 19107, USA.
Thiyagarajan
Manimekalai M
MM
Takida
Satoshi
S
Wedegaertner
Philip B
PB
D000107:Acetylation; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000068438:Brimonidine Tartrate; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019308:Palmitic Acid / Q000378:metabolism; D054799:Phospholipase C beta; D011485:Protein Binding; D011810:Quinoxalines / Q000494:pharmacology; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D000316:Adrenergic alpha-Agonists; C118981:G-protein Beta gamma; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007295:Inositol Phosphates; D007527:Isoenzymes; D011810:Quinoxalines; D018341:Receptors, Adrenergic, alpha-2; D019308:Palmitic Acid; D000068438:Brimonidine Tartrate; D000242:Cyclic AMP; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins