RGS4 binds to membranes through an amphipathic alpha -helix.
RGS4, a mammalian GTPase-activating protein for G protein alpha subunits, requires its N-terminal 33 amino acids for plasma membrane localization and biological activity (Srinivasa, S. P., Bernstein, L. S., Blumer, K. J., and Linder, M. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5584-5589). In this study, we tested the hypothesis that the N-terminal domain mediates membrane binding by forming an amphipathic alpha-helix. RGS4 bound to liposomes containing anionic phospholipids in a manner dependent on the first 33 amino acids. Circular dichroism spectroscopy of a peptide corresponding to amino acids 1-31 of RGS4 revealed that the peptide adopted an alpha-helical conformation in the presence of anionic phospholipids. Point mutations that either neutralized positive charges on the hydrophilic face or substituted polar residues on the hydrophobic face of the model helix disrupted plasma membrane targeting and biological activity of RGS4 expressed in yeast. Recombinant mutant proteins were active as GTPase-activating proteins in solution but exhibited diminished binding to anionic liposomes. Peptides corresponding to mutants with the most pronounced phenotypes were also defective in forming an alpha-helix as measured by circular dichroism spectroscopy. These results support a model for direct interaction of RGS4 with membranes through hydrophobic and electrostatic interactions of an N-terminal alpha-helix.
Bernstein
L S
LS
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Grillo
A A
AA
Loranger
S S
SS
Linder
M E
ME
D000595:Amino Acid Sequence; D000838:Anions / Q000378:metabolism; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011506:Proteins; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*
D000838:Anions; D008081:Liposomes; D008563:Membrane Lipids; D011506:Proteins; D020710:RGS Proteins; C107413:RGS16 protein; C105346:Rgs5 protein, mouse; C098799:RGS4 protein