Abstract
Numerous studies have attested to the importance of the extreme C terminus of G protein α 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 Gαq 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 Gαq proteins in which glycine was replaced with various amino acids and the C-terminal five Gαq residues with the corresponding Gαi or Gαs sequence. Coupling properties of the mutated Gαq proteins were determined after coexpression with a panel of 13 Gi-and Gs -selective receptors and compared with those of Gα proteins modified in only one module. Gα proteins modified in both modules are significantly more efficacious in channeling non-Gq -selective receptors to Gq-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 Gα 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 Gα proteins and that distinct determinants of selectivity exist for individual receptors.
Footnotes
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doi:10.1124/jpet.104.080424.
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ABBREVIATIONS: GPCR, G protein-coupled receptor; KOR, κ opioid receptor; GLP1, glucagon like peptide 1; GIP, gastric inhibitory peptide; NPY4, neuropeptide Y4; MCH-1R, melanin concentrating hormone-1 receptor; SSTR1, somatostatin type I receptor; HBSS, Hank's balanced salt solution; HEK, human embryonic kidney; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; BSA, bovine serum albumin; GTPγS, guanosine 5′-O-(3-thio)triphosphate; PLCβ, phospholipase Cβ; AlF –4, fluoroaluminate; PTX, pertussis toxin; U50,488, (trans)-3,4-dichloro-N-methyl-N[lqsb]2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide.
- Received November 9, 2004.
- Accepted December 17, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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