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.

Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands

Abstract: CCL19 and CCL21 are endogenous agonists for the seven-transmembrane receptor CCR7. They are equally active in promoting G protein stimulation and chemotaxis. Yet, we find that they result in striking differences in activation of the G protein-coupled receptor kinase (GRK)/ss-arrestin system. CCL19 leads to robust CCR7 phosphorylation and beta-arrestin2 recruitment catalyzed by both GRK3 and GRK6 whereas CCL21 activates GRK6 alone. This differential GRK activation leads to distinct functional consequences. Although each ligand leads to beta-arrestin2 recruitment, only CCL19 leads to redistribution of beta-arrestin2-GFP into endocytic vesicles and classical receptor desensitization. In contrast, these agonists are both capable of signaling through GRK6 and beta-arrestin2 to ERK kinases. Thus, this mechanism for "ligand bias" whereby endogenous agonists activate different GRK isoforms leads to functionally distinct pools of beta-arrestin.

Distinct conformations of GPCR–β-arrestin complexes mediate desensitization, signaling, and endocytosis

Abstract: β-Arrestins (βarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR–βarr complexes: the “tail” conformation, with βarr primarily coupled to the phosphorylated GPCR C-terminal tail, and the “core” conformation, where, in addition to the phosphorylated C-terminal tail, βarr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of βarrs is unknown. Here, we created a mutant form of βarr lacking the “finger-loop” region, which is unable to form the core conformation but retains the ability to form the tail conformation. We find that the tail conformation preserves the ability to mediate receptor internalization and βarr signaling but not desensitization of G protein signaling. Thus, the two GPCR–βarr conformations can carry out distinct functions.

Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation

Abstract: G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and β-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the β2-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for β2AR in the presence of Nb80 compared to the affinity of isoprenaline for β2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.

Reciprocal In Vivo Regulation of Myocardial G Protein–Coupled Receptor Kinase Expression by β-Adrenergic Receptor Stimulation and Blockade

Abstract: Background —Impaired myocardial β-adrenergic receptor (βAR) signaling, including desensitization and functional uncoupling, is a characteristic of congestive heart failure. A contributing mechanism for this impairment may involve enhanced myocardial β-adrenergic receptor kinase (βARK1) activity because levels of this βAR-desensitizing G protein–coupled receptor kinase (GRK) are increased in heart failure. An hypothesis has emerged that increased sympathetic nervous system activity associated with heart failure might be the initial stimulus for βAR signaling alterations, including desensitization. We have chronically treated mice with drugs that either activate or antagonize βARs to study the dynamic relationship between βAR activation and myocardial levels of βARK1. Methods and Results —Long-term in vivo stimulation of βARs results in the impairment of cardiac βAR signaling and increases the level of expression (mRNA and protein) and activity of βARK1 but not that of GRK5, a second GRK abundantly expressed in the myocardium. Long-term β-blocker treatment, including the use of carvedilol, improves myocardial βAR signaling and reduces βARK1 levels in a specific and dose-dependent manner. Identical results were obtained in vitro in cultured cells, demonstrating that the regulation of GRK expression is directly linked to βAR signaling. Conclusions —This report demonstrates, for the first time, that βAR stimulation can significantly increase the expression of βARK1, whereas β-blockade decreases expression. This reciprocal regulation of βARK1 documents a novel mechanism of ligand-induced βAR regulation and provides important insights into the potential mechanisms responsible for the effectiveness of β-blockers, such as carvedilol, in the treatment of heart failure.

Catecholamines, cardiac beta-adrenergic receptors, and heart failure.

Abstract: It is now generally accepted that chronically elevated stimulation of the cardiac b-adrenergic system is toxic to the heart and that such stimulation may contribute to the pathogenesis of congestive heart failure of various causes. Administration of either b-adrenergic agonists or phosphodiesterase inhibitors has been shown to decrease survival of patients with chronic heart failure, even though they produce immediate and long-term hemodynamic benefits. 1 Moreover, in human heart failure, as well as in several animal models, elevated circulating catecholamines lead, via various compensatory mechanisms, to decreased levels and functional activity of cardiac b1-adrenergic receptors (b1ARs) and thus to marked desensitization of the heart to inotropic b-adrenergic stimulation.2

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)