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Mapping the putative G protein-coupled receptor (GPCR) docking site on GPCR kinase 2: insights from intact cell phosphorylation and recruitment assays.

ABSTRACT: G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and ?-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (?N), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the ?N and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated ?(2A)-adrenergic receptors (?(2A)ARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to G?? and ?(2A)AR with EC50 values of 15 nM and 8 ?M, respectively. We show that mutations in ?N (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu(4), Val(7), Leu(8), Val(11), and Ser(12), directly interacts with receptors, whereas residues such as Asp(10), Tyr(13), Ala(16), Met(17), Gly(475), Val(477), and Ile(485) are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity.

SUBMITTER: Beautrait A

PROVIDER: S-EPMC4155688 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Mapping the putative G protein-coupled receptor (GPCR) docking site on GPCR kinase 2: insights from intact cell phosphorylation and recruitment assays.

Beautrait Alexandre A Michalski Kevin R KR Lopez Thomas S TS Mannix Katelynn M KM McDonald Devin J DJ Cutter Amber R AR Medina Christopher B CB Hebert Aaron M AM Francis Charnelle J CJ Bouvier Michel M Tesmer John J G JJ Sterne-Marr Rachel R

The Journal of biological chemistry 20140721 36

G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further ...[more]

PMID: 25049229

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Project description:Mutations in the BRCA2 tumor suppressor protein leave individuals susceptible to breast, ovarian and other cancers. The BRCA2 protein is a critical component of the DNA repair pathways in eukaryotes, and also plays an integral role in fostering genomic variability through meiotic recombination. Although present in many eukaryotes, as a whole the BRCA2 gene is weakly conserved. Conserved fragments of 30 amino acids (BRC repeats), which mediate interactions with the recombinase RAD51, helped detect orthologs of this protein in other organisms. The carboxy-terminal of the human BRCA2 has been shown to be phosphorylated by checkpoint kinases (Chk1/Chk2) at T3387, which regulate the sequestration of RAD51 on DNA damage. However, apart from three BRC repeats, the Drosophila melanogaster gene has not been annotated and associated with other functionally relevant sequence fragments in human BRCA2. In the current work, the carboxy-terminal phosphorylation threonine site (E=9.1e-4) and a new BRC repeat (E=17e-4) in D. melanogaster has been identified, using a fragmented alignment methodology (FRAGAL). In a similar study, FRAGAL has also identified a novel half-a- tetratricopeptide (HAT) motif (E=11e-4), a helical repeat motif implicated in various aspects of RNA metabolism, in Utp6 from yeast. The characteristic three aromatic residues with conserved spacing are observed in this new HAT repeat, further strengthening my claim. The reference and target sequences are sliced into overlapping fragments of equal parameterized lengths. All pairs of fragments in the reference and target proteins are aligned, and the gap penalties are adjusted to discourage gaps in the middle of the alignment. The results of the best matches are sorted based on differing criteria to aid the detection of known and putative sequences. The source code for FRAGAL results on these sequences is available at https://github.com/sanchak/FragalCode, while the database can be accessed at www.sanchak.com/fragal.html.

Dataset Information

Mapping the putative G protein-coupled receptor (GPCR) docking site on GPCR kinase 2: insights from intact cell phosphorylation and recruitment assays.

Publications

Mapping the putative G protein-coupled receptor (GPCR) docking site on GPCR kinase 2: insights from intact cell phosphorylation and recruitment assays.

Similar Datasets

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