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.

Production of adrenergic receptors in yeast.

Abstract: Using a recombinant yeast strain expressing human beta 2 adrenergic receptors under a galactose-inducible promoter, we established conditions for receptor production in 1-15 liter fermenter culture. Crucial factors contributing to consistent high-level expression included the use of selective glucose-free medium, the maintenance of the pH of the culture at 7.2-7.5 and the presence of an antagonist. The expression strategy and production conditions used with the beta 2 adrenergic receptor were then employed to express the human alpha 2-C2 adrenergic receptor in Saccharomyces cerevisiae. Galactose-induced yeast cells displayed specific, high-affinity [3H]rauwolscine binding and contained a 50-kDa species recognized by an alpha 2-C2 receptor specific antiserum. In fermenter culture, up to 10(5) high-affinity [3H]rauwolscine binding sites per cell (corresponding to 30-60 pmol/mg of protein) were obtained. The high expression level combined with relative ease and low cost of scaling-up make yeast a promising alternative to mammalian cells for the production of adrenergic and other G-protein-coupled receptors for structural studies.

Temperature immutability of adenyl cyclase-coupled β adrenergic recptors

Abstract: OBSERVATIONS from several laboratories have suggested that the well established classification of adrenergic receptors into α and β subtypes is not immutable. Several groups have reported that in isolated perfused frog hearts, stimulation of cardiac rate and contractility by catecholamines has the properties of a classical β adrenergic response when experiments are performed at warm temperatures (25°–37° C) but of an α adrenergic response when experiments are performed at cold temperatures (5°–15° C)1–3. At warm temperatures, the order of potency of agonists in stimulating these preparations—isoproterenol>adrenaline>noradrenaline—is classical for a β adrenergic receptor. Similarly, effects of the catecholamines at warm temperatures are blocked by propranolol but not by the α adrenergic antagonist phentolamine. When the same experiments are performed at temperatures below 25° C, the order of potency of agonists is reversed to that characteristic of α adrenergic receptors. Also at lower temperatures α adrenergic antagonists such as phenoxybenzamine and phentolamine block the effects of adrenaline, whereas β adrenergic antagonists such as propranolol are ineffective. Gradations of response can be achieved by varying the temperature between 37° C and 10° C. Similar observations have been reported for the rat heart1. Also in a dog heart–lung bypass preparation the β receptors seem to become ineffective at 15° C, whereas α receptors retain their effectiveness4. On the basis of such observations, Kunos et al.3 proposed that α and β adrenergic receptors may represent allosteric configurations of the same receptor macromolecule which could be modulated by among other factors, temperature.

Affinity chromatography of human platelet α2-adrenergic receptors

Abstract: Catecholamines, such as epinephrine, inhibit the enzyme adenylate cyclase (EC 4.6.1.1) via a specific receptor mechanism involving α2-adrenergic receptors. In order to facilitate purification of these inhibitory receptors we have prepared a highly effective biospecific affinity adsorbent. The immobilized ligand SKF 101253 is a 3-benzazepine with α2-adrenergic antagonist activity. SKF 101253 is coupled to Sepharose CL-4B by using a bifunctional reagent (1,4-butanediol diglycidyl ether) which also provides a hydrophilic spacer moiety between the ligand and the gel matrix. Membranes from human platelets, containing α2-adrenergic receptors, can be specifically labeled with [3H]yohimbine and can be solubilized with digitonin without loss of their α2-adrenergic binding characteristics. Chromatography of solubilized human platelet membrane preparations on the SKF 101253-Sepharose CL-4B affinity gel results in the adsorption of 70-80% of the initial [3H]yohimbine binding activity. Adsorption to the affinity gel is blocked by both α-adrenergic antagonists (phentolamine ≥ yohimbine > prazosin) and by α-adrenergic agonists [p-aminoclonidine > (-)-epinephrine > (+)-epinephrine]. Similarly, elution of specific [3H]yohimbine binding activity from the affinity gel is effected with the aforementioned agonists and antagonists in the same order of potency. Other drugs that do not interact appreciably with α-adrenergic receptors, such as (-)-isoproterenol, (-)-alprenolol, atropine, and carbachol, are ineffective for both the blockade of adsorption and the elution of specific [3H]yohimbine binding activity from the affinity gel. In addition to the specificity of the interaction, chromatography of solubilized human platelet membrane preparations on the SKF 101253-Sepharose CL-4B affinity gel results in a 40-50% overall yield and an approximately 200-fold increase in the specific binding activity for [3H]yohimbine. The results indicate that the SKF 101253-Sepharose CL-4B affinity adsorbent should provide a powerful tool for the purification of the adenylate cyclase-inhibitory α2-adrenergic receptor of human platelets.

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)