Robert J. Lefkowitz

Bio: Robert J. Lefkowitz is an academic researcher from Howard Hughes Medical Institute. The author has contributed to research in topics: Receptor & G protein-coupled receptor. The author has an hindex of 214, co-authored 860 publications receiving 147995 citations. Previous affiliations of Robert J. Lefkowitz include University of Nice Sophia Antipolis & University of Stuttgart.

Desensitization, internalization, and signaling functions of β-arrestins demonstrated by RNA interference

Abstract: β-Arrestins bind to activated G protein-coupled receptor kinase-phosphorylated receptors, which leads to their desensitization with respect to G proteins, internalization via clathrin-coated pits, and signaling via a growing list of “scaffolded” pathways. To facilitate the discovery of novel adaptor and signaling roles of β-arrestins, we have developed and validated a generally applicable interfering RNA approach for selectively suppressing β-arrestins 1 or 2 expression by up to 95%. β-Arrestin depletion in HEK293 cells leads to enhanced cAMP generation in response to β2-adrenergic receptor stimulation, markedly reduced β2-adrenergic receptor and angiotensin II receptor internalization and impaired activation of the MAP kinases ERK 1 and 2 by angiotensin II. This approach should allow discovery of novel signaling and regulatory roles for the β-arrestins in many seven-membrane-spanning receptor systems.

Agonist-dependent phosphorylation of the mouse delta-opioid receptor: involvement of G protein-coupled receptor kinases but not protein kinase C.

Abstract: With chronic opiate use, opioid receptor desensitization may be one of the important mechanisms underlying the development of opiate tolerance and addiction. Opioid receptors belong to the G protein-coupled receptor superfamily. In this study, the mouse delta-opioid receptor (delta OR) was used in a model system to investigate the role of opioid receptor phosphorylation in receptor desensitization. When expressed in 293 cells and exposed to agonist, the delta OR underwent receptor-specific desensitization within 10 min. This agonist-induced desensitization corresponded temporally to a 3-fold increase in receptor phosphorylation. Phorbol ester, but not forskolin, also stimulated phosphorylation of the delta OR in 293 cells. Although down-regulation of protein kinase C failed to affect agonist-induced receptor phosphorylation, it abolished phorbol ester-induced receptor phosphorylation. Agonist-induced delta OR phosphorylation must therefore involve kinases other than protein kinase C. Whereas overexpression of a dominant negative mutant (K220R) of beta-adrenergic receptor kinase-1 (beta ARK1) in 293 cells significantly reduced agonist-dependent phosphorylation of the delta OR, overexpression of beta ARK1 or G protein-coupled receptor kinase-5 significantly enhanced this phosphorylation. Concordantly, beta ARK1-K220R overexpression reduced agonist-dependent delta OR desensitization, whereas beta ARK1 overexpression enhanced this densensitization. We conclude that short term desensitization of the delta OR involves phosphorylation of the receptor by one or more G protein-coupled receptor kinases.

Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors

Abstract: Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the α2A adrenergic receptor (α2A AR), with ERK activation mediated by the β2 adrenergic receptor (β2 AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the α2A AR, like that mediated by both the β2 AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant β-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both α2A and β2 AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the β2 AR, the α2A AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although β2 AR stimulation leads to redistribution of both the β2 AR and EGFR, activation of the α2A AR leads to redistribution of the EGFR but the α2A AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.

Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase is associated with phosphorylation of the beta-adrenergic receptor

Abstract: Preincubation of turkey erythrocytes with catecholamines desensitizes the beta-adrenergic receptor-adenylate cyclase complex in the plasma membranes of these cells. Photoaffinity labeling of the beta-adrenergic receptors with 125I-labeled p-azidobenzylcarazolol (125I-pABC) and subsequent analysis by NaDodSO4/polyacrylamide gel electrophoresis demonstrates an altered mobility of receptor peptides from desensitized cells compared to controls [Stadel, J.M., Nambi, P., Lavin, T.N., Heald, S.L., Caron, M.G. & Lefkowitz, R.J. (1982) J. Biol. Chem. 257, 9242-9245]. The time course of alteration in beta-adrenergic receptor mobility correlates with that for desensitization of isoproterenol-stimulated adenylate cyclase activity. The altered mobility of the receptor peptides from desensitized cells is also observed if the receptors are first purified and then photoaffinity labeled with 125I-pABC. The cyclic nucleotide analog 8-bromoadenosine 3',5'-cyclic monophosphate partially mimics catecholamines in promoting desensitization of the adenylate cyclase and modification of the receptor. Phosphorylation of the beta-adrenergic receptor in intact turkey erythrocytes was assessed by preincubating the cells with [32P]orthophosphate, desensitizing them with catecholamine, purifying the receptors, and then subjecting them to NaDodSO4/polyacrylamide gel electrophoresis. Desensitization is associated with a 2- to 3-fold increase in 32P incorporation into the receptor, which also demonstrates the characteristic alterations in mobility. These effects are blocked by the beta-adrenergic antagonist propranolol. Purified turkey erythrocyte beta-adrenergic receptors could be phosphorylated by incubation with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase. The mobility of the phosphorylated receptor peptides on NaDodSO4/polyacrylamide gel electrophoresis appears to correspond to that of the desensitized receptors. These data show that catecholamine-induced desensitization of adenylate cyclase in turkey erythrocytes correlates with a stable modification of the beta-adrenergic receptor and is associated with agonist-promoted phosphorylation of beta-receptor peptides.

Role of acidic amino acids in peptide substrates of the beta-adrenergic receptor kinase and rhodopsin kinase.

Abstract: The beta-adrenergic receptor kinase (beta-ARK) phosphorylates G protein coupled receptors in an agonist-dependent manner. Since the exact sites of receptor phosphorylation by beta-ARK are poorly defined, the identification of substrate amino acids that are critical to phosphorylation by the kinase are also unknown. In this study, a peptide whose sequence is present in a portion of the third intracellular loop region of the human platelet alpha 2-adrenergic receptor is shown to serve as a substrate for beta-ARK. Removal of the negatively charged amino acids surrounding a cluster of serines in this alpha 2-peptide resulted in a complete loss of phosphorylation by the kinase. A family of peptides was synthesized to further study the role of acidic amino acids in peptide substrates of beta-ARK. By kinetic analyses of the phosphorylation reactions, beta-ARK exhibited a marked preference for negatively charged amino acids localized to the NH2-terminal side of a serine or threonine residue. While there were no significant differences between glutamic and aspartic acid residues, serine-containing peptides were 4-fold better substrates than threonine. Comparing a variety of kinases, only rhodopsin kinase and casein kinase II exhibited significant phosphorylation of the acidic peptides. Unlike beta-ARK, RK preferred acid residues localized to the carboxyl-terminal side of the serine. A feature common to beta-ARK and RK was a much greater Km for peptide substrates as compared to that for intact receptor substrates.

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Abstract: Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression Here we investigate the requirements for structure and delivery of the interfering RNA To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference The effects of this interference were evident in both the injected animals and their progeny Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process

How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions.

Abstract: The secretion of glucocorticoids (GCs) is a classic endocrine response to stress. Despite that, it remains controversial as to what purpose GCs serve at such times. One view, stretching back to the time of Hans Selye, posits that GCs help mediate the ongoing or pending stress response, either via basal levels of GCs permitting other facets of the stress response to emerge efficaciously, and/or by stress levels of GCs actively stimulating the stress response. In contrast, a revisionist viewpoint posits that GCs suppress the stress response, preventing it from being pathologically overactivated. In this review, we consider recent findings regarding GC action and, based on them, generate criteria for determining whether a particular GC action permits, stimulates, or suppresses an ongoing stressresponse or, as an additional category, is preparative for a subsequent stressor. We apply these GC actions to the realms of cardiovascular function, fluid volume and hemorrhage, immunity and inflammation, metabolism, neurobiology, and reproductive physiology. We find that GC actions fall into markedly different categories, depending on the physiological endpoint in question, with evidence for mediating effects in some cases, and suppressive or preparative in others. We then attempt to assimilate these heterogeneous GC actions into a physiological whole. (Endocrine Reviews 21: 55‐ 89, 2000)