Copper and zinc inhibit Galphas function: a nucleotide-free state of Galphas induced by Cu2+ and Zn2+.
The stimulatory GTP-binding protein of adenylyl cyclase (AC) regulates hormone-stimulated production of cAMP. Here, we demonstrate that Cu(2+) and Zn(2+) inhibit the steady-state GTPase activity of the alpha subunit of GTP-binding protein (Galpha(s)) but do not alter its intrinsic GTPase activity. Cu(2+) and Zn(2+) decrease steady-state GTPase activity by inhibiting the binding of GTP to Galpha(s). Moreover, Cu(2+) and Zn(2+) increase GDP dissociation from Galpha(s) and render the G protein in a nucleotide-free state. However, these cations do not alter the dissociation of the guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) that is already bound to the Galpha(s). Because of their ability to inhibit GTPgammaS binding, preincubation of Cu(2+) or Zn(2+) with Galpha(s) does not permit GTPgammaS to activate Galpha(s) and stimulate AC activity. However, preincubation of Galpha(s) with GTPgammaS followed by addition of Cu(2+) or Zn(2+) did not alter the ability of Galpha(s) to stimulate AC activity. Interestingly, AlF(4)(-) partially restored the ability of Galpha(s), which had been preincubated with Cu(2+) or Zn(2+), to stimulate AC; AlF(4)(-) does not permit the re-association of unbound GDP with Galpha(s). Thus, the interaction of AlF(4)(-) with the nucleotide-free Galpha(s) is sufficient to activate AC. Using antibodies to the N and C termini of Galpha(s), we show that the Cu(2+) interaction site on the G protein is in the C terminus. We conclude that Cu(2+) and Zn(2+) generate a nucleotide-free state of Galpha(s) and that, in the absence of any nucleotide, the gamma-phosphate mimic of GTP, AlF(4)(-), alters Galpha(s) structure sufficiently to permit stimulation of AC activity. Moreover, our finding that isoproterenol-stimulated AC activity was more sensitive to inhibition by Cu(2+) and Zn(2+) as compared with forskolin-stimulated activity is consistent with Galpha(s) being a primary target of these cations in regulating the signaling from receptor to AC.
Gao
Xianlong
X
Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois 60153, USA.
Du
Ziyun
Z
Patel
Tarun B
TB
D000262:Adenylyl Cyclases / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002412:Cations; D002417:Cattle; D005576:Colforsin / Q000494:pharmacology; D003300:Copper / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D007545:Isoproterenol / Q000494:pharmacology; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors; D015032:Zinc / Q000737:chemistry / Q000494:pharmacology*
D000318:Adrenergic beta-Agonists; D002412:Cations; D009711:Nucleotides; D006153:Guanosine Diphosphate; D005576:Colforsin; D003300:Copper; D006861:Hydrogen Peroxide; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D015032:Zinc; D007545:Isoproterenol