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CELLULAR AND MOLECULAR

A Highly Conserved Glycine within Linker I and the Extreme C Terminus of G Protein Subunits Interact Cooperatively in Switching G Protein-Coupled Receptor-to-Effector Specificity

7TM Pharma, Hoersholm, Denmark (E.K., L.M., P.K.N., R.J.); Ernest Gallo Clinic and Research Center, University of California, San Francisco, Emeryville, California (L.M., M.W., J.L.W.); Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland (J.E., G.M.); and Laboratory for Molecular Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark (A.H., M.M.R.)

Numerous studies have attested to the importance of the extreme C terminus of G protein subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor recognition by G_q proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant G_q proteins in which glycine was replaced with various amino acids and the C-terminal five G_q residues with the corresponding G_i or G_s sequence. Coupling properties of the mutated G_q proteins were determined after coexpression with a panel of 13 G_i-and G_s -selective receptors and compared with those of G proteins modified in only one module. G proteins modified in both modules are significantly more efficacious in channeling non-G_q -selective receptors to G_q-mediated signaling events compare with those containing each module alone. Additive effects of both modules were observed even if individual modules lacked an effect on GPCR-to-effector specificity. Dually modified G proteins were also superior in conferring high-affinity agonist sites onto a coexpressed GPCR in the absence, but not in the presence, of guanine nucleotides. Together, our data suggest that receptor-G protein coupling selectivity involves cooperative interactions between the extreme C terminus and linker I of G proteins and that distinct determinants of selectivity exist for individual receptors.

Address correspondence to: Dr. Evi Kostenis, 7TM Pharma, Fremtidsvej 3, 2970 Hoersholm, Denmark. E-mail: ek{at}7tm.com

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