title: A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity.
abstract: Numerous studies have attested to the importance of the extreme C terminus of G protein alpha 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 Galpha(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 Galpha(q) proteins in which glycine was replaced with various amino acids and the C-terminal five Galpha(q) residues with the corresponding Galpha(i) or Galpha(s) sequence. Coupling properties of the mutated Galpha(q) proteins were determined after coexpression with a panel of 13 G(i)-and G(s) -selective receptors and compared with those of Galpha proteins modified in only one module. Galpha 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 Galpha 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 Galpha proteins and that distinct determinants of selectivity exist for individual receptors.
authors.affiliation: 7TM Pharma, Fremtidsvej 3, 2970 Hoersholm, Denmark. ek@7tm.com
authors.forename: Evi
authors.initials: E
authors.lastname: Kostenis
authors.forename: Lene
authors.initials: L
authors.lastname: Martini
authors.forename: James
authors.initials: J
authors.lastname: Ellis
authors.forename: Maria
authors.initials: M
authors.lastname: Waldhoer
authors.forename: Arne
authors.initials: A
authors.lastname: Heydorn
authors.forename: Mette M
authors.initials: MM
authors.lastname: Rosenkilde
authors.forename: Pia K
authors.initials: PK
authors.lastname: Norregaard
authors.forename: Rasmus
authors.initials: R
authors.lastname: Jorgensen
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Whistler
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D007295:Inositol Phosphates; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004247:DNA; D002118:Calcium; D005998:Glycine