title: Identification of structural features in the G-protein regulatory motif required for regulation of heterotrimeric G-proteins.
abstract: The G-protein regulatory (GPR) motif, a conserved 25-30 amino acid domain found in multiple mammalian proteins, stabilizes the GDP-bound conformation of Galpha(i), inhibits guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to Galpha(i) and competes for Gbetagamma binding to Galpha. To define the core GPR motif and key amino acid residues within a GPR peptide (TMGEEDFFDLLAKSQSKRMDDQRVDLAG), we determined the effect of truncation, insertion, and alanine substitutions on peptide-mediated inhibition of GTPgammaS binding to purified Galpha(i1). The bioactive core GPR peptide consists of 17 amino acids ((7)F-R(23)). Within this core motif, two hydrophobic sectors ((7)FF(8) and (10)LL(11)) and Q(22) are required for bioactivity, whereas M19A and R23A increased IC(50) values by 70-fold. Disruption of spatial relationships between the required sectors in the amino and carboxyl regions of the peptide also resulted in a loss of biological activity. Mutation of three charged sectors ((4)EED(6), R(18), (20)DD(21)) within the 28-amino acid GPR decreased peptide affinity by approximately 10-fold. Alanine substitutions of selected residues within the core GPR peptide differently influenced peptide inhibition of GTPgammaS binding to Galpha(i) versus Galpha(o). These data provide a platform for the development of novel, G-protein-selective therapeutics that inhibit Galpha(i)- mediated signaling, selectively activate Gbetagamma-sensitive effectors, and/or disrupt specific regulatory input to G-proteins mediated by GPR-containing proteins.
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 1901 Perdido Street, New Orleans, LA 70118, USA.
authors.forename: Yuri K
authors.initials: YK
authors.lastname: Peterson
authors.forename: Starr
authors.initials: S
authors.lastname: Hazard
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Graber
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Lanier
mesh_terms: D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D007313:Insecta; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D000596:Amino Acids; D010455:Peptides; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; D008274:Magnesium; D000409:Alanine