ArticleProceedings of the National Academy of Sciences of the United States of America1994
An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G beta gamma subunits.
Article in Proceedings of the National Academy of Sciences of the United States of America, 1994. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 46 citations in OpenAlex.
- Essential strategies for the detection of constitutive and ligand-dependent Gi-directed activity of 7TM receptors using bioluminescence resonance energy transfer.bioRxiv : the preprint server for biology · 2024Article
- Structural studies of phosphorylation-dependent interactions between the V2R receptor and arrestin-2.Nature communications · 2021Article
- GPCR drug discovery: integrating solution NMR data with crystal and cryo-EM structures.Nature reviews. Drug discovery · 2019Review
- Phosphorylation-induced conformation of βNature communications · 2018Article
- G-protein-coupled receptor kinase 2 and endothelial dysfunction: molecular insights and pathophysiological mechanisms.Journal of smooth muscle research = Nihon Heikatsukin Gakkai kikanshi · 2015Review
- Neuroendocrine-immune interactions and responses to exercise.Sports medicine (Auckland, N.Z.) · 2011Review
- G-protein coupled receptor kinases as modulators of G-protein signalling.The Journal of physiology · 1999Review
- Phosphorylation-independent inhibition of parathyroid hormone receptor signaling by G protein-coupled receptor kinases.Proceedings of the National Academy of Sciences of the United States of America · 1999Article
- Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases.Proceedings of the National Academy of Sciences of the United States of America · 1997Article
- Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.Proceedings of the National Academy of Sciences of the United States of America · 1996Article
- The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.Proceedings of the National Academy of Sciences of the United States of America · 1995Article
- Lack of association of G-protein beta 2- and gamma 2-subunit N-terminal fragments provides evidence against the coiled-coil model of subunit-beta gamma assembly.The Biochemical journal · 1995Article
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Authors and funding
4 authors at 2 institutions in 1 country.
Funding
Abstract
Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.