G protein-coupled receptor (GPCR) desensitization and internalization are dependent on phosphorylation by G protein coupled receptor kinases (GRKs). In order to better understand this process we sought to further characterize GRK function in cells.
Through mutagenesis of the GRK2 PH domain, specific residues in the loop-1 region were identified as phospholipid binding determinants, whereas residues distal to the carboxyl-terminal amphipathic α-helix and one residue in β-sheet-3 were identified as G βγ binding determinants. Two mutants, GRK2(K567E/R578E) and GRK2(R587Q), were characterized as significantly and selectively disrupted in phospholipid and G βγ interaction, respectively. Furthermore, in vivo studies of these mutants demonstrated that both the G βγ and phospholipid binding activities are indispensable for efficient GRK2 function in cells.
Through efforts to identify novel GRK interacting proteins, we found that GRK2 and GRK5 could both bind and stoichiometrically phosphorylate the cytoskeletal protein tubulin both in vitro and in vivo . In addition, GRK-phosphorylated tubulin was found to preferentially associate with the microtubule fraction during in vitro assembly assays suggesting potential functional significance.
Based on identification of consensus caveolin binding sequences in GRKs we tested their direct binding to caveolin in vitro and discovered specific caveolin interactions were mediated by GRK1, GRK2 and GRK5. Binding was shown to depend on specific amino-terminal (GRK1-6)- and PH domain (GRK2/3)-localized caveolin binding and to take place in vivo . Functional significance for the GRK/caveolin interaction was demonstrated by the potent inhibition of GRK-mediated phosphorylation of both receptor and peptide substrates by caveolin-1 and -3 scaffolding domain peptides.
Finally, we discovered a specific ability of both GRK2 and GRK3 to bind G αq/11 both in vitro and in vivo in an activation-dependent manner. GRK2 demonstrated weak GAP activity toward G αq , yet effectively inhibited G αq -mediated activation of PLC-β both in vitro and in cells, possibly through sequestration of activated G αq .
Taken together, these data have significantly expanded our knowledge of GRK function in cells. In addition, they have revealed diverse roles for GRKs that are certain to impact the way they function in GPCR regulation.
