Vol. 298, Issue 2, 840-847, August 2001

Monovalent Anions Differentially Modulate Coupling of the ₂-Adrenoceptor to G_s Splice Variants

Roland Seifert

Department of Pharmacology and Toxicology, The University of Kansas, Lawrence, Kansas

The ₂-adrenoceptor (₂AR) fused to the long splice variant of G_s (G_sL), but not the ₂AR fused to the short splice variant of G_s (G_sS) shows the hallmarks of high constitutive activity, i.e., strong activation of adenylyl cyclase (AC) by GTP and strong inhibition of AC by inverse agonist. These coupling differences are the result of differences in GDP affinity of G_s splice variants. The aim of this study was to identify experimental variables that differentially affect ₂AR coupling to G_sS and G_sL. NaCl substantially reduced agonist-independent AC activation by GTP and inverse agonist inhibition and enhanced agonist stimulation of AC in Sf9 insect cell membranes expressing the ₂AR-G_sL fusion protein. Salts reduced inverse agonist inhibition and increased agonist stimulation of AC in the order of efficiency NaI ~ KI > NaBr ~ KBr > NaCl ~ LiCl ~ KCl ~ RbCl ~ CsCl ~ choline chloride, indicating that monovalent anions determine salt effects. Salts inhibited guanosine 5'-O-(3-thiotriphosphate)-mediated AC activation by G_sL without ₂AR in the order of efficiency NaI > NaBr > NaCl. NaCl enhanced the affinity of G_sL for GDP. Salts had much smaller effects on ₂AR ligand regulation of AC in membranes expressing ₂AR-G_sS than in membranes expressing ₂AR-G_sL. These data are explained by a model in which anions increase the GDP affinity of G_sL more efficiently than the GDP affinity of G_sS, and, thereby, decrease the efficiency of the agonist-free ₂AR and increase the efficiency of the agonist-occupied ₂AR at promoting GDP dissociation from G_sL. Thus, monovalent anions differentially regulate ₂AR-coupling to G_sS and G_sL.