Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha.
Agonist-bound receptors activate heterotrimeric (alpha beta gamma) G proteins by catalysing replacement of GDP bound to the alpha-subunit by GTP. mutations in the C terminus of the alpha-subunit, its covalent modification by pertussis toxin-catalysed ribosylation of ADP, peptide-specific antibodies directed against it, and peptides mimicking C-terminal sequences, all inhibit receptor-mediated activation of G proteins. The logical prediction--that specific amino-acid residues at the C-termini of alpha-subunits can determine the abilities of individual G proteins to discriminate among specific subsets of receptors--has so far not been tested experimentally. Different hormone receptors specifically activate Gq or Gi, whose alpha-subunits (alpha q or alpha i) stimulate phosphatidylinositol-specific phospholipase C or inhibit adenylyl cyclase, respectively. Here we replace C-terminal amino acids of alpha q with the corresponding residues of alpha i2 to create alpha q/alpha i2 chimaeras that can mediate stimulation of phospholipase C by receptors otherwise coupled exclusively to Gi. A minimum of three alpha i2 amino acids, including a glycine three residues from the C terminus, suffices to switch the receptor specificity of the alpha q/alpha i2 chimaeras. We propose that a C-terminal turn, centered on this glycine, plays an important part in specifying receptor interactions of G proteins in the Gi/Go/Gz family.
Department of Pharmacology, University of California, San Francisco 94143.
B R
BR
Conklin
Z
Z
Farfel
K D
KD
Lustig
D
D
Julius
H R
HR
Bourne
D037361:Adenylate Cyclase Toxin; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000596:Amino Acids; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D005998:Glycine