LOCALIZATION OF THE SITES FOR CA2-BINDING PROTEINS ON G-PROTEIN-COUPLED RECEPTOR KINASES()

Inhibition of G protein-coupled receptor kinases (GRKs) by Ca2+-bindin g proteins has recently emerged as a general mechanism of GRK regulati on. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-sp ecific Ca2+-binding protein recoverin, other GRKs can be inhibited by Ca2+-calmodulin. To dissect the mechanism of this inhibition at the mo lecular level, we localized the GRK domains involved in Ca2+-binding p rotein interaction using a series of GST-GRK fusion proteins. GRK1, GR K2, and GRK5, which represent the three known GRK subclasses, were eac h found to possess two distinct calmodulin-binding sites. These sites were localized to the N- and C-terminal regulatory regions within doma ins rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin had no clear ly defined structural characteristics. interestingly: while the recove rin and calmodulin-binding sites in GRK1 do not overlap, recoverin-GRK 1 interaction is inhibited by calmodulin, most likely via an allosteri c mechanism. Further analysis of the individual calmodulin sites in GR K5 suggests that the C-terminal site plays the major role in GRK5-calm odulin interaction. While specific mutation within the N-terminal site had no effect on calmodulin-mediated inhibition of GRK5 activity, del etion of the C-terminal site attenuated the effect of calmodulin on GR K5. and the simultaneous mutation of both sites rendered the enzyme ca lmodulin-insensitive. These studies provide new insight into the mecha nism of Ca2+-dependent regulation of GRKs.