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A Key Serine for the GTPase-Activating Protein Function of Regulator of G Protein Signaling Proteins Is Not a General Target for 14-3-3 Interactions

Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom

Mammalian regulator of G protein signaling (RGS) proteins are highly conserved within the RGS domain. Of amino acids that are universal, a serine residue at the C terminus of this domain has been described as the binding site in RGS7 for 14-3-3 proteins. However, studies with the related RGS3 indicate that the site of interaction is not within the RGS domain. We confirm that the interaction of RGS3 with 14-3-3 and 14-3-3 requires Ser²⁶⁴ and not the RGS domain and show both that mutation of the conserved RGS domain serine, Ser⁴⁹⁶ in RGS3, to either alanine or aspartate does not prevent binding of 14-3-3 proteins and that 14-3-3 proteins do not inhibit GTPase-activating protein (GAP) activity against receptor-activated G_o1. However, mutation of Ser⁴⁹⁶ does directly impair the action of RGS3 as a GAP against receptor-activated G_o1. We mutated the equivalent serine residue in the family B/R4 RGS proteins RGS1 and RGS16. Using two distinct assay formats, conversion to aspartate virtually abolished GAP activity, whereas conversion to alanine decreased potency 20-fold. Neither alteration modulated interactions with 14-3-3 or 14-3-3, but the 14-3-3 proteins did not modulate the GAP activity of the wild-type or mutant RGS proteins. Although interactions between 14-3-3 proteins and many RGS proteins can be observed, this does not involve this conserved serine and does not inherently modify GAP function.

Address correspondence to: Dr. Graeme Milligan, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. E-mail: g.milligan{at}bio.gla.ac.uk

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