GRK2 activation by receptors: role of the kinase large lobe and carboxyl-terminal tail.

G protein-coupled receptor (GPCR) kinases (GRKs) were discovered by virtue of their ability to phosphorylate activated GPCRs. They constitute a branch of the AGC kinase superfamily, but their mechanism of activation is largely unknown. To initiate a study of GRK2 activation, we sought to identify sites on GRK2 remote from the active site that are involved in interactions with their substrate receptors. Using the atomic structure of GRK2 in complex with Gbetagamma as a guide, we predicted that residues on the surface of the kinase domain that face the cell membrane would interact with the intracellular loops and carboxyl-terminal tail of the GPCR. Our study focused on two regions: the kinase large lobe and an extension of the kinase domain known as the C-tail. Residues in the GRK2 large lobe whose side chains are solvent exposed and facing the membrane were targeted for mutagenesis. Residues in the C-tail of GRK2, although not ordered in the crystal structure, were also targeted because this region has been implicated in receptor binding and in the regulation of AGC kinase activity. Four substitutions out of 20, all within or adjacent to the C-tail, resulted in significant deficiencies in the ability of the enzyme to phosphorylate two different GPCRS: rhodopsin, and the beta(2)-adrenergic receptor. The mutant exhibiting the most dramatic impairment, V477D, also showed significant defects in phosphorylation of nonreceptor substrates. Interestingly, Michaelis-Menten kinetics suggested that V477D had a 12-fold lower k(cat), but no changes in K(M), suggesting a defect in acquisition or stabilization of the closed state of the kinase domain. V477D was also resistant to activation by agonist-treated beta(2)AR. Therefore, Val477 and other residues in the C-tail are expected to play a role in the activation of GRK2 by GPCRs.

Self-masking in an intact ERM-merlin protein: an active role for the central alpha-helical domain.

Ezrin/radixin/moesin (ERM) family members provide a regulated link between the cortical actin cytoskeleton and the plasma membrane to govern membrane structure and organization. Here, we report the crystal structure of intact insect moesin, revealing that its essential yet previously uncharacterized alpha-helical domain forms extensive interactions with conserved surfaces of the band four-point-one/ezrin/radixin/moesin (FERM) domain. These interdomain contacts provide a functional explanation for how PIP(2) binding and tyrosine phosphorylation of ezrin lead to activation, and provide an understanding of previously enigmatic loss-of-function missense mutations in the tumor suppressor merlin. Sequence conservation and biochemical results indicate that this structure represents a complete model for the closed state of all ERM-merlin proteins, wherein the central alpha-helical domain is an active participant in an extensive set of inhibitory interactions that can be unmasked, in a rheostat-like manner, by coincident regulatory factors that help determine cell polarity and membrane structure.