Copper and Zinc Inhibit Gαs Function

• Published in: The Journal of biological chemistry Vol. 280; no. 4; pp. 2579 - 2586
• Main Authors: Gao, Xianlong, Du, Ziyun, Patel, Tarun B.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 28-01-2005
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M409791200

The stimulatory GTP-binding protein of adenylyl cyclase (AC) regulates hormone-stimulated production of cAMP. Here, we demonstrate that Cu2+ and Zn2+ inhibit the steady-state GTPase activity of the α subunit of GTP-binding protein (Gαs) but do not alter its intrinsic GTPase activity. Cu2+ and Zn2+ decrease steady-state GTPase activity by inhibiting the binding of GTP to Gαs. Moreover, Cu2+ and Zn2+ increase GDP dissociation from Gαs 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 (GTPγS) that is already bound to the Gαs. Because of their ability to inhibit GTPγS binding, preincubation of Cu2+ or Zn2+ with Gαs does not permit GTPγS to activate Gαs and stimulate AC activity. However, preincubation of Gαs with GTPγS followed by addition of Cu2+ or Zn2+ did not alter the ability of Gαs to stimulate AC activity. Interestingly, AlF4− partially restored the ability of Gαs, which had been preincubated with Cu2+ or Zn2+, to stimulate AC; AlF4− does not permit the re-association of unbound GDP with Gαs. Thus, the interaction of AlF4− with the nucleotide-free Gαs is sufficient to activate AC. Using antibodies to the N and C termini of Gαs, we show that the Cu2+ interaction site on the G protein is in the C terminus. We conclude that Cu2+ and Zn2+ generate a nucleotide-free state of Gαs and that, in the absence of any nucleotide, the γ-phosphate mimic of GTP, AlF4−, alters Gαs structure sufficiently to permit stimulation of AC activity. Moreover, our finding that isoproterenol-stimulated AC activity was more sensitive to inhibition by Cu2+ and Zn2+ as compared with forskolin-stimulated activity is consistent with Gαs being a primary target of these cations in regulating the signaling from receptor to AC.