Activation of G Protein-Coupled Receptor Kinase 1 Involves Interactions between Its N-Terminal Region and Its Kinase Domain
- 25 January 2011
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 50 (11), 1940-1949
- https://doi.org/10.1021/bi101606e
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.Keywords
This publication has 38 references indexed in Scilit:
- Molecular basis for activation of G protein-coupled receptor kinasesThe EMBO Journal, 2010
- Role of the Amino Terminus of G Protein-Coupled Receptor Kinase 2 in Receptor PhosphorylationBiochemistry, 2009
- GRK2 Activation by Receptors: Role of the Kinase Large Lobe and Carboxyl-Terminal TailBiochemistry, 2009
- Different Properties of the Native and Reconstituted Heterotrimeric G Protein TransducinBiochemistry, 2008
- Structures of Rhodopsin Kinase in Different Ligand States Reveal Key Elements Involved in G Protein-coupled Receptor Kinase ActivationPublished by Elsevier BV ,2008
- Identification and Characterization of Disulfide Bonds in Proteins and Peptides from Tandem MS Data by Use of the MassMatrix MS/MS Search EngineJournal of Proteome Research, 2007
- Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylationThe EMBO Journal, 2007
- The hallmark of AGC kinase functional divergence is its C-terminal tail, a cis-acting regulatory moduleProceedings of the National Academy of Sciences, 2007
- Arrestin can act as a regulator of rhodopsin photochemistryVision Research, 2006
- Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanismNature, 2006