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.

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1994-04-26

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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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Lefkowitz

Robert J. Lefkowitz

The Chancellor's Distinguished Professor of Medicine

Dr. Lefkowitz’s memoir, A Funny Thing Happened on the Way to Stockholm, recounts his early career as a cardiologist and his transition to biochemistry, which led to his Nobel Prize win.

Robert J. Lefkowitz, M.D. is Chancellor’s Distinguished Professor of Medicine and Professor of Biochemistry and Chemistry at the Duke University Medical Center. He has been an Investigator of the Howard Hughes Medical Institute since 1976. Dr. Lefkowitz began his research career in the late 1960’s and early 1970’s when there was not a clear consensus that specific receptors for drugs and hormones even existed. His group spent 15 difficult years developing techniques for labeling the receptors with radioactive drugs and then purifying the four different receptors that were known and thought to exist for adrenaline, so called adrenergic receptors. In 1986 Dr. Lefkowitz transformed the understanding of what had by then become known as G protein coupled receptors because of the way the receptor signal for the inside of a cell through G proteins, when he and his colleagues cloned the gene for the beta2-adrenergic receptor. They immediately recognized the similarity to a molecule called rhodopsin which is essentially a light receptor in the retina. This unexpected finding established the beta receptor and rhodopsin as the first member of a new family of proteins. Because each has a peptide structure, which weaves across the cell membrane seven times, these receptors are referred to as seven transmembrane receptors. This super family is now known to be the largest, most diverse and most therapeutically accessible of all the different kinds of cellular receptors. There are almost a thousand members of this receptor family and they regulate virtually all known physiological processes in humans. They include the receptors not only to numerous hormones and neurotransmitters but for the receptors which mediate the senses of sweet and bitter taste and smell amongst many others. Dr. Lefkowitz also discovered the mechanism by which receptor signaling is turned off, a process known as desensitization. Dr. Lefkowitz work was performed at the most fundamental and basic end of the research spectrum and has had remarkable consequences for clinical medicine. Today, more than half of all prescription drug sales are of drugs that target either directly or indirectly the receptors discovered by Dr. Lefkowitz and his trainees. These include amongst many others beta blockers, angiotensin receptor blockers or ARBs and antihistamines. Over the past decade he has discovered novel mechanisms by which the receptors function which may lead to the development of an entirely new class of drugs called “biased agonists”. Several such compounds are already in advanced stages of clinical testing. Dr. Lefkowitz has received numerous honors and awards, including the National Medal of Science, the Shaw Prize, the Albany Prize, and the 2012 Nobel Prize in Chemistry. He was elected to the USA National Academy of Sciences in 1988, the Institute of Medicine in 1994, and the American Academy of Arts and Sciences in 1988.


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