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Abstract
G protein-coupled receptor kinases (GRKs) are a family of serine/threonine kinases that phosphorylate many activated G protein-coupled receptors (GPCRs) and play an important role in GPCR desensitization. Our previous work has demonstrated that the C-terminal conserved region (CC) of GRK-2 participates in interaction with rhodopsin and that this interaction is necessary for GRK-2-mediated receptor phosphorylation (Gan, X. Q., Wang, J. Y., Yang, Q. H., Li, Z., Liu, F., Pei, G., and Li, L. (2000) J. Biol. Chem. 275, 8469-8474). In this report, we further investigated whether the CC of other GRKs had the same functions and defined the specific sequences in CC that are required for the functions. The CC regions of GRK-1, GRK-2, and GRK-5, representatives of the three subfamilies of GRKs, could bind rhodopsin in vitro and inhibit GRK-2-mediated phosphorylation of rhodopsin, but not a peptide GRK substrate. Through a series of mutagenesis analyses, a proline-rich motif in the CC was identified as the key element involved in the interaction between the CC region and rhodopsin. Point mutations of this motif not only disrupted the interaction of GRK-2 with rhodopsin but also abolished the ability of GRK-2 to phosphorylate rhodopsin. The findings that the CC region of GRKs interact only with the light-activated but not the non-activated rhodopsin and that the N-terminal domain of GRK-2 interacts with rhodopsin in a light-independent manner suggest that the CC region is responsible for the recognition of activated GPCRs in the canonical model.
Highlights
G protein-coupled receptor kinases (GRKs)1 constitute a family of seven serine/threonine protein kinases that recognize and phosphorylate agonist-bound, or activated, G protein-coupled receptors (GPCRs) [1, 2]
We have reported previously that the C-terminal conserved region (CC) region of GRK-2 can inhibit GRK-2-mediated phosphorylation of rhodopsin in vitro through direct binding of this region to rhodopsin and that a mutant of GRK-2 with CC deletion resulted in a complete loss of its ability to phosphorylate rhodopsin, demonstrating that interaction of the CC region with rhodopsin is required for GRK-2 to phosphorylate the receptor [29]
The results demonstrated that the CC of both GRK-1 and GRK-5, like CC of GRK-2, could inhibit the phosphorylation of rhodopsin by GRK-2
Summary
G protein-coupled receptor kinases (GRKs) constitute a family of seven serine/threonine protein kinases that recognize and phosphorylate agonist-bound, or activated, G protein-coupled receptors (GPCRs) [1, 2]. GRKs interact with many proteins, including G␣ [4, 15, 16] and G␥ [17] subunits, clathrin [18], GRK-interacting protein GIT1 [19], caveolin-1 [20], phosphoinositide 3-kinase-␣ and -␥ [21], cytoskeletal protein tubulin and actin [6, 22, 23], and Raf kinase inhibitor protein [24] Many of these interactions are thought to be important for regulating the localization and enzymatic activity of GRKs. In the canonical model, GRKs rapidly recognize and phosphorylate activated GPCRs, leading to their homologous desensitization. The present work reveals a common role of this conserved region of GRKs in substrate recognition and defines a proline-rich motif within this region required for this interaction
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