GDP Affinity and Order State of the Catalytic Site Are Critical for Function of Xanthine Nucleotide-selective Galpha s Proteins

doi:10.1074/jbc.M210162200

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GDP Affinity and Order State of the Catalytic Site Are Critical for Function of Xanthine Nucleotide-selective G $alpha$ _s Proteins^*

Andreas Gille, Katharina Wenzel-Seifert, Michael B. Doughty^§^¶, and Roland Seifert

From the Departments of Pharmacology and Toxicology and ^§ Medicinal Chemistry, The University of Kansas, Lawrence, Kansas 66045-7582 and ^¶ Department of Chemistry and Physics, Southeastern Louisiana University, Hammond, Louisiana 70402-0878

Xanthine nucleotide-selective small GTP-binding proteins with an Asp/Asn mutation are valuable for the analysis of individualGTP-binding proteins in complex systems. Similar applicationscan be devised for heterotrimeric G-proteins. However, Asp/Asnmutants of G $alpha$ _o, G $alpha$ ₁₁, and G $alpha$ ₁₆ were inactive. An additional Gln/Leumutation in the catalytic site, reducing GTPase activity and increasingGDP affinity, was required to generate xanthine nucleotide-selectiveunspecified G-protein $alpha$ -subunit (G $alpha$ ). Our study aim was to generatexanthine nucleotide-selective mutants of G $alpha$ _s, the stimulatoryG-protein of adenylyl cyclase. The short splice variant of G $alpha$ _s(G $alpha$ _sS) possesses higher GDP affinity than the long splice variant(G $alpha$ _sL). Nucleoside 5'-[ $gamma$ -thio]triphosphates (NTP $gamma$ Ss) and nucleoside5'-[ $beta$ , $gamma$ -imido]triphosphates effectively activated a G $alpha$ _sS mutantwith a D280N exchange (G $alpha$ _sS-N280), whereas nucleotides activateda G $alpha$ _sL mutant with a D295N exchange (G $alpha$ _sL-N295) only weakly. TheGln/Leu mutation enhanced G $alpha$ _sL-N295 activity. NTP $gamma$ Ss activatedG $alpha$ _sS-N280 and a G $alpha$ _sL mutant with a Q227L and D295N exchange (G $alpha$ _sL-L227/N295)with similar potencies, whereas xanthosine 5'-triphosphate andxanthosine 5'-[ $beta$ , $gamma$ -imido]triphosphate were more potent than GTPand guanosine 5'-[ $beta$ , $gamma$ -imido]triphosphate, respectively. G $alpha$ _sS-N280interacted with the $beta$ ₂-adrenoreceptor and exhibited high-affinityXTPase activity. Collectively, (i) G $alpha$ _sS-N280 is the first functionalxanthine nucleotide-selective G $alpha$ with the Asp/Asn mutation alone;(ii) sufficiently high GDP affinity is crucial for G $alpha$ Asp/Asnmutant function; (iii) with nucleoside 5'-triphosphates and nucleoside5'-[ $beta$ , $gamma$ -imido]triphosphates, G $alpha$ _s-N280 and G $alpha$ _sL-L227/N295 exhibitxanthine nucleotide selectivity, whereas NTP $gamma$ Ss sterically perturbthe catalytic site of G $alpha$ and annihilate xanthineselectivity.

^* This work was supported by Grant 0051404Z of the Heartland Affiliate of The American Heart Association (to R. S.), the J.R. & Inez Jay Biomedical Research Award of The University of Kansas(to R. S.), and a predoctoral fellowship of the Studienstiftungdes Deutschen Volkes (to A. G.).The costs of publication of thisarticle were defrayed in part by the payment of page charges.The article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

This paper is in remembrance of the 12^th anniversary of the reunification of Germany on October 3^rd, 2002, without which this project would not have beenconducted.

To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, The University of Kansas, Malott Hall, Rm.5064, 1251 Wescoe Hall Dr., Lawrence, KS 66045-7582. Tel.: 785-864-3525;Fax: 785-864-5219; E-mail: rseifert@ku.edu.

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GDP Affinity and Order State of the Catalytic Site Are Critical for Function of Xanthine Nucleotide-selective Gs Proteins*

GDP Affinity and Order State of the Catalytic Site Are Critical for Function of Xanthine Nucleotide-selective G $alpha$ _s Proteins^*