GRK2 Activation by Receptors: Role of the Kinase Large Lobe and Carboxyl-Terminal Tail

Abstract

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 Gβγ 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 (C) 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. Conserved 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 twenty, all within or adjacent to the C-tail, resulted in significant deficiencies in the ability of the enzyme to phosphorylate activated GPCRs, rhodopsin and the β 2 -adrenergic receptor (β 2 AR). The mutant exhibiting the most dramatic impairment, V477D, also showed significant defects in phosphorylation of non-receptor substrates. Interestingly, Michaelis-Menten kinetics suggested that V477D had 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 β 2 AR. Therefore, Val477 and other residues in the C-tail are expected to play a role in the activation of GRK2 by GPCRs.G protein-coupled receptors (GPCRs), distinguished by their seven transmembrane helices and their ability to couple to heterotrimeric G proteins (Gαβγ), are activated by hormones, neurotransmitters and sensory signals, and regulate many diverse processes. Receptor stimulation catalyzes the binding of GTP to the Gα subunit, allowing Gα and Gβγ to directly † This work was funded by the National Science Foundation grants MCB0315888 and MCB0744739 (to RSM), National Institutes of Health grants HL086865 and HL071818 and American Heart Association Scientist Development Grant 0235273N (to JJGT), and a summer undergraduate research fellowship from the Northeast Affiliate of the American Heart Association (to EDB). *To whom correspondence should be addressed. Biology Department, Siena College, Morrell Science Center, 515 Loudon Road, Loudonville, NY 12211, Tel: (518) 783-2462; Fax: (518) ; email: sternemarr@siena.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 May 26. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript modulate effectors such as adenylyl cyclase, phospholipase Cβ, cGMP phosphodiesterase, ion channels and ...

Role for the Regulator of G-Protein Signaling Homology Domain of G Protein-Coupled Receptor Kinases 5 and 6 in β2-Adrenergic Receptor and Rhodopsin Phosphorylation

“…1G. Although in the present study these residues were not further examined, ET revealed interesting features consistent with results from the mutagenesis studies outlined above (Huang et al, 2009;Sterne-Marr et al, 2009). …”

“…It was proposed these sites may be involved either directly in the allosteric GPCR activation or in intramolecular interactions of the N terminus with residues surrounding the catalytic site that stabilize the active state (Huang et al, 2009;Pao et al, 2009;Sterne-Marr et al, 2009). Pao et al have also shown that a GRK2 peptide mimicking the N-terminal residues 1 to 14 noncompetitively inhibited GRK2 phosphorylation of GPCRs (Pao et al, 2009), most consistent with interference with the proposed intramolecular mechanism.…”

“…It was also suggested that the dimer interface of GRK6 is a likely area of protein-protein interactions (Lodowski et al, 2006), and that residues 5 to 30 of the N terminus and the C-terminal extension of the kinase domain proximal to the hinge region are possible sites for allosteric receptor binding (Singh et al, 2008). It has been found that mutations in the GRK2 kinase domain, notably V477D, impair its phosphorylation of rhodopsin and ␤2AR (Sterne-Marr et al, 2009). In addition, a number of mutations in the kinase small and large lobes of GRK1 (R191A/K, Y274A, V476A, and V484A) were found to block rhodopsin phosphorylation (Huang et al, 2009), and it was proposed that a cooperative interaction between the N terminus and the kinase domain was important for GPCR activation.…”

Role of Helix 8 of the Thyrotropin-Releasing Hormone Receptor in Phosphorylation by G Protein-Coupled Receptor Kinase

“…Three different regions of the ␤2-adrenergic receptor tail show little apparent affinity for GRK2, but the proximal end of helix 8 has not been tested (Benovic et al, 1990). The structures of GRK2 alone and in complexes with G␤␥ and G␣ q have been solved (Lodowski et al, 2003;Tesmer et al, 2005;Singh et al, 2008;Sterne-Marr et al, 2009), and GRK2 seems to be capable of interacting simultaneously with G␤␥, G␣ q , and receptor. Molecular modeling suggests that one molecule of GRK2 may interact with a GPCR dimer (Tesmer et al, 2005;Singh et al, 2008).…”

“…Using a combination of crystallographic, mutagenic, and kinetic analyses, Tesmer and coworkers (Singh et al, 2008;Huang et al, 2009;Sterne-Marr et al, 2009) identified a surface in the aminoterminal region and kinase domains of GRKs 1 and 2 that is critical for kinase interaction with activated GPCRs. The cytoplasmic tail of the receptor sits in an open groove on the kinase while different regions of the receptor interact elsewhere with the enzyme.…”