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Biochemistry. 2011 Mar 22;50(11):1940-9. doi: 10.1021/bi101606e. Epub 2011 Feb 22.

Activation of G protein-coupled receptor kinase 1 involves interactions between its N-terminal region and its kinase domain.

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1
Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109-2216, United States.

Abstract

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its ∼20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.

PMID:
21265573
PMCID:
PMC3069497
DOI:
10.1021/bi101606e
[Indexed for MEDLINE]
Free PMC Article
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