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R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 199 records · Page 11Linked to original sources
PubMed1993

Constitutively active mutants of the alpha 2-adrenergic receptor.

We have mutated a single residue, Thr373 [corrected], in the C-terminal portion of the third intracellular loop of the alpha 2C10-adrenergic receptor into five different amino acids. In analogy with the effect of similar mutations in the alpha 1B- and beta 2-adrenergic receptors, these substitutions resulted in two major biochemical modifications: 1) increased constitutive activity of the alpha 2-adrenergic receptor leading to agonist-independent inhibition of adenylyl cyclase and 2) increased affinity of the receptor for binding agonist but not antagonists. The increased constitutive activity of the mutated alpha 2-adrenergic receptors could be inhibited by pertussis toxin, clearly indicating that it results from spontaneous ligand-independent receptor coupling to Gi. In contrast, the increased affinity of the mutant receptors for binding agonists was unaffected by pertussis toxin treatment, indicating that this is an inherent property of the receptors not dependent on interaction with Gi. Coexpression of the receptor mutants with the receptor-specific kinase, beta ARK1, indicated that the constitutively active alpha 2-adrenergic receptors are substrates for beta-adrenergic receptor kinase (beta ARK)-mediated phosphorylation even in the absence of agonist. These findings strengthen the idea that constitutively active adrenergic receptors mimic the "active" state of a G protein-coupled receptor adopting conformations similar to those induced by agonist when it binds to wild type receptors. In addition, these results extend the notion that in the adrenergic receptor family the C-terminal portion of the third intracellular loop plays a general role in the processes involved in receptor activation.

Adenylate Cyclase Toxin↗
PubMed1993

The substance P receptor, which couples to Gq/11, is a substrate of beta-adrenergic receptor kinase 1 and 2.

The agonist-occupied forms of several G-protein-coupled receptors that modulate the activity of adenylycyclase via Gs (e.g. beta 2-adrenergic) or Gi (e.g. alpha 2-adrenergic and cardiac muscarinic) are phosphorylated by beta-adrenergic receptor kinases (beta ARK 1 and beta ARK 2). beta ARK-catalyzed phosphorylation of these receptors appears to correlate with their agonist-induced desensitization. The possibility that beta ARK isozymes may also be involved in the desensitization of other G-protein-coupled receptors such as those mediating phosphoinositide (PI) hydrolysis was tested by determining the phosphorylation of the substance P receptor (SPR), which is coupled to PI hydrolysis in numerous tissues. Rat SPR was expressed in Sf9 cells, partially purified, and reconstituted in phospholipid vesicles. The reconstituted SPR bound the SPR agonist substance P, 125I-labeled with Bolton-Hunter reagent, with low affinity. However, addition of purified Gq/11 to the reconstituted SPR resulted in the conversion of all the receptors to a high affinity state, suggesting that SPR couples to Gq/11. Phosphorylation of the reconstituted SPR with purified beta ARK 1 or 2 in the absence and presence of substance P (SP) was then studied. In the presence of 100 microM SP, both kinases promoted phosphorylation of the receptor to a stoichiometry of 9 +/- 2 mol of phosphate/mol of receptor. However, no phosphorylation of the receptor could be detected in the absence of agonist. Agonist-induced phosphorylation of the receptor was blocked by coincubation with the SPR antagonist spantide. These results show that beta ARK isozymes may regulate the function of both adenylylcyclase as well as PI-coupled receptors, and suggest a role for beta ARK isozymes in SPR signal transduction.

Animals↗
PubMed1993

Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.

Adenylyl Cyclases↗
PubMed1993

The binding site for the beta gamma subunits of heterotrimeric G proteins on the beta-adrenergic receptor kinase.

The beta gamma subunits of heterotrimeric G proteins play important roles in regulating receptor-stimulated signal transduction processes. Recently appreciated among these is their role in the signaling events that lead to the phosphorylation and subsequent desensitization of muscarinic cholinergic (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267, 2222-2227) and beta-adrenergic (Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267) receptors. Beta gamma mediates the membrane targeting of the beta-adrenergic receptor kinase (beta ARK), in response to receptor activation, through a specific beta ARK-beta gamma interaction. This process utilizes the membrane-anchoring properties of the isoprenylated gamma subunit of beta gamma. In the present study, we have employed three distinct approaches to identify the region within the carboxyl terminus of beta ARK which binds beta gamma and thereby results in membrane translocation. We studied the ability of beta gamma to enhance the enzymatic activity of a series of truncated mutants of bovine beta ARK1, the ability of glutathione S-transferase fusion proteins containing various lengths of the carboxyl terminus of beta ARK to bind beta gamma subunits, and the ability of synthetic peptides comprised of beta ARK sequences to inhibit beta gamma activation of beta ARK1. We find that the minimal beta gamma binding domain of beta ARK is localized to a 125-amino acid residue stretch, the distal end of which is located 19 residues from the carboxyl terminus. A single 28-mer peptide (Trp643 to Ser670) derived from this sequence effectively inhibited beta gamma activation of beta ARK1, with an IC50 of 76 microM. The identification of this "beta gamma binding domain" on beta ARK and the development of peptide inhibitors provide important tools for the study of G protein-coupled receptor desensitization, as well as for the investigation of beta gamma activation of other G protein-effector systems.

Animals↗
PubMed1993

Coupling of the expressed alpha 1B-adrenergic receptor to the phospholipase C pathway in Xenopus oocytes. The role of Go.

alpha 1B-Adrenergic receptor mRNA was injected into Xenopus oocytes, resulting in a norepinephrine-evoked Cl- current. The response was proportional to norepinephrine concentration, blocked by prazosin, and dependent on intracellular Ca2+ derived from inositol trisphosphate-sensitive stores. Oocytes treated with 2 micrograms/ml pertussis toxin showed a time-dependent decrease of the norepinephrine response, taking up to 72 h to show an 80% decrease. Overnight treatment with 10 micrograms/ml pertussis toxin also resulted in 80% reduction. Responses to two other cloned receptors (M1-muscarinic and serotonin-1c) expressed in oocytes were also reduced 50% or more by 72 h of pertussis toxin treatment. Pertussis toxin labeling of the cloned Xenopus alpha o-subunit translated in vitro showed that it was a significantly poorer substrate for pertussis toxin than the two mammalian alpha o-subunits expressed and assayed under identical conditions. This unexpected biochemical behavior of the Xenopus alpha o-subunit is in agreement with the rather unusual treatment conditions required to observe the effects of pertussis toxin on the receptor-evoked Cl- current in the oocyte. Injection of mammalian heterotrimeric G(o) but not Gi3 significantly enhanced the norepinephrine-evoked Cl- current in oocytes. Injection of mixtures of anti-sense oligonucleotides to the Xenopus alpha o-subunit reduced the norepinephrine-evoked Cl- current by 60% within 24 h, compared with oocytes injected with the oligonucleotides encoding sense sequences. These studies indicate that the expressed alpha 1B-adrenergic receptor, like the native muscarinic receptor, utilizes G(o) to couple to the phospholipase C-mediated Cl- current in Xenopus oocytes.

Animals↗
PubMed1993

Antagonism of catecholamine receptor signaling by expression of cytoplasmic domains of the receptors.

The actions of many hormones and neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins). These receptors are characterized by a highly conserved topographical arrangement in which seven transmembrane domains are connected by intracellular and extracellular loops. The interaction between these receptors and G proteins is mediated in large part by the third intracellular loop of the receptor. Coexpression of the third intracellular loop of the alpha 1B-adrenergic receptor with its parent receptor inhibited receptor-mediated activation of phospholipase C. The inhibition extended to the closely related alpha 1C-adrenergic receptor subtype, but not the phospholipase C-coupled M1 muscarinic acetylcholine receptor nor the adenylate cyclase-coupled D1A dopamine receptor. These results suggest that the receptor-G protein interface may represent a target for receptor antagonist drugs.

Amino Acid Sequence↗
PubMed1993

A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model.

We have replaced the C-terminal portion of the third intracellular loop of the beta 2-adrenergic receptor (residues 266-272) with the homologous region of the alpha 1B-adrenergic receptor. In a fashion analogous to the reciprocal mutations of the alpha 1B receptor previously described (Cotecchia, S., Exum, S., Caron, M. G., and Lefkowitz, R. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2896-2900), this conservative substitution leads to agonist-independent activation of adenylyl cyclase. In addition, the constitutively active mutant receptor exhibits: (i) an increased affinity for agonists (even in the absence of guanine nucleotide-binding regulatory protein (G protein)) but not antagonists, with the extent of affinity increase being correlated with the intrinsic activity of the ligand; (ii) an increased potency of agonists for stimulation of adenylyl cyclase; and (iii) an increased intrinsic activity of partial agonists. We document that our experimental findings with the mutant receptor cannot be adequately rationalized within the theoretical framework of the Ternary Complex Model (De Lean, A., Stadel, J. M., and Lefkowitz, R. J. (1980) J. Biol. Chem. 255, 7108-7117) which postulates that receptor activation requires the agonist-promoted formation of an active, "ternary" complex of agonist, receptor, and G protein. We show, through extensive computer simulations, that an extended version of this model that includes an explicit isomerization of the receptor (R) to an active state (R*) closely models all our findings for both the mutant and the wild-type receptors. Study of such constitutively active mutant G protein-coupled receptors should help elucidate the molecular nature of the processes involved in receptor activation.

Adenylyl Cyclases↗
PubMed1993

Cloning and sequence analysis of the human beta 1-adrenergic receptor 5'-flanking promoter region.

We present 3.1 kb of the nucleotide sequence from the 5'-flanking region of the human beta 1-adrenergic receptor gene. The first 1.0 kb upstream from the translational start site is composed of 72% G + C residues. The sequence was analyzed for the presence of transcriptional regulatory elements and contains putative thyroid hormone, glucocorticoid hormone and cAMP response elements. These putative hormone response elements support physiological evidence that thyroid and glucocorticoid hormones regulate beta 1AR function by affecting receptor expression in tissues such as heart and adipose, where beta 1-adrenergic receptors are important regulators of heart rate and lipolysis, respectively.

Base Sequence↗
PubMed1993

A beta-adrenergic receptor kinase-like enzyme is involved in olfactory signal termination.

We have previously shown that second-messenger-dependent kinases (cAMP-dependent kinase, protein kinase C) in the olfactory system are essential in terminating second-messenger signaling in response to odorants. We now document that subtype 2 of the beta-adrenergic receptor kinase (beta ARK) is also involved in this process. By using subtype-specific antibodies to beta ARK-1 and beta ARK-2, we show that beta ARK-2 is preferentially expressed in the olfactory epithelium in contrast to findings in most other tissues. Heparin, an inhibitor of beta ARK, as well as anti-beta ARK-2 antibodies, (i) completely prevents the rapid decline of second-messenger signals (desensitization) that follows odorant stimulation and (ii) strongly inhibits odorant-induced phosphorylation of olfactory ciliary proteins. In contrast, beta ARK-1 antibodies are without effect. Inhibitors of protein kinase A and protein kinase C also block odorant-induced desensitization and phosphorylation. These data suggest that a sequential interplay of second-messenger-dependent and receptor-specific kinases is functionally involved in olfactory desensitization.

Animals↗
PubMed1993

Overexpression of beta-arrestin and beta-adrenergic receptor kinase augment desensitization of beta 2-adrenergic receptors.

Receptor-specific or homologous desensitization of beta 2-adrenergic receptors is thought to be effected via phosphorylation of the receptor by the beta-adrenergic receptor kinase (beta ARK), followed by binding of beta-arrestin. We have generated stably transfected Chinese hamster ovary cell lines overexpressing either of the two regulatory proteins and also expressing low or high levels of beta 2-adrenergic receptors (approximately 80 and approximately 600 fmol/mg of membrane protein). In these cells, we studied the process of desensitization induced by the beta-adrenergic receptor agonist isoproterenol. In cells expressing high levels of beta 2-adrenergic receptors, desensitization to high concentrations of isoproterenol (previously shown to be mediated by both beta ARK and protein kinase A) amounted to approximately 50% in control cells, approximately 80% in beta ARK-overexpressing cells, and approximately 90% in beta-arrestin-overexpressing cells. In cells expressing low levels of beta 2-adrenergic receptors, these values were approximately 50, approximately 60, and approximately 60%, respectively. Desensitization to low concentrations of isoproterenol (previously shown to be essentially protein kinase A-mediated and not receptor-specific, i.e. heterologous) was not affected by overexpression of either beta ARK or beta-arrestin. These data suggest that in cells expressing high levels of beta 2-adrenergic receptors, beta-arrestin and beta ARK become limiting for homologous receptor desensitization. They provide further support for the involvement of these two proteins in the regulation of beta 2-adrenergic receptor function.

Adenylyl Cyclases↗
PubMed1993

Beta-adrenergic receptor kinase-2 and beta-arrestin-2 as mediators of odorant-induced desensitization.

beta-Adrenergic receptor kinase (beta ARK) and beta-arrestin function in the homologous or agonist-activated desensitization of G protein-coupled receptors. The isoforms beta ARK-2 and beta-arrestin-2 are highly enriched in and localized to the dendritic knobs and cilia of the olfactory receptor neurons where the initial events of olfactory signal transduction occur. Odorants induce a rapid and transient elevation of adenosine 3',5'-monophosphate (cAMP), which activates a nonspecific cation channel and produces membrane depolarization. Preincubation of rat olfactory cilia with antibodies raised against beta ARK-2 and beta-arrestin-2 increased the odorant-induced elevation of cAMP and attenuated desensitization. These results suggest that beta ARK-2 and beta-arrestin-2 mediate agonist-dependent desensitization in olfaction.

Animals↗
PubMed1993

Beta-adrenergic receptor sequestration. A potential mechanism of receptor resensitization.

Continuous exposure of cells to hormonal agonists often causes a rapid waning of the stimulated response. This desensitization effect has been extensively studied in the beta-adrenergic receptor system, and attributed largely to the rapid phosphorylation of the receptor by two kinases. Over a similar time frame (seconds to minutes), agonists also trigger a selective loss in the capacity of receptors to bind hydrophilic but not hydrophobic ligands, a phenomenon termed sequestration. There is some evidence suggesting that sequestration represents the rapid internalization of receptors, but the functional significance of sequestration has remained unclear. Upon the removal of agonist, both desensitization and sequestration are readily reversed with similar kinetics (t1/2 approximately 3 min for both). To investigate the possibility that receptor sequestration is involved in this resensitization of the adenylyl cyclase response, we applied two distinct approaches to block receptor sequestration: by pretreating cells with sucrose and by creating a sequestration-defective beta 2-adrenergic receptor by site-specific mutagenesis. Both approaches effectively disabled receptor sequestration, with little effect on adenylyl cyclase stimulation or on desensitization. However, in both cases, no recovery from desensitization was apparent even 20 min after the removal of agonist. Similarly, pretreating cells with concanavalin A almost completely blocked receptor sequestration and resensitization but only partially inhibited other receptor functions. Our results therefore suggest that sequestration of beta 2-adrenergic receptors is a mechanism involved in reactivating and recycling desensitized receptors.

Adenylyl Cyclases↗
PubMed1993

Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins.

Adrenoceptors are prototypic members of the superfamily of seven transmembrane domain, G protein-coupled receptors. Study of the properties of several mutationally activated adrenoceptors is deepening understanding of the normal functioning of this ubiquitous class of receptors. The new findings suggest an expansion of the classical ternary complex model of receptor action to include an explicit isomerization of the receptors from an inactive to an active state which couples to the G protein ('allosteric ternary complex model'). This isomerization involves conformational changes which may occur spontaneously, or be induced by agonists or appropriate mutations which abrogate the normal 'constraining' function of the receptor, allowing it to 'relax' into the active conformation. Robert Lefkowitz and colleagues discuss the physiological and pathophysiological implications of these new insights into regulation of receptor activity.

Amino Acid Sequence↗
PubMed1993

Identification, quantification, and localization of mRNA for three distinct alpha 1 adrenergic receptor subtypes in human prostate.

The dynamic component of bladder outlet obstruction caused by benign prostatic hyperplasia (BPH) is regulated by alpha 1 adrenergic receptors (alpha 1-AR) located in the prostatic stroma. Recently two alpha 1-AR subtypes (alpha 1A, alpha 1B) have been identified in the human prostate by both functional and pharmacological assays. However, the presence of the alpha 1C subtype has not been evaluated, presumably due to the lack of availability of selective ligands for this receptor subtype. We have used molecular techniques to investigate the mRNA expression of all three alpha 1-AR subtypes in the human prostate. RNA extracted from the prostate gland of 15 patients was used in ribonuclease protection assays to identify the expression of three alpha 1-AR subtype mRNAs. Quantitative solution hybridization assays further identify the predominant subtype of alpha 1-AR mRNA to be the alpha 1C, which represents approximately 70% of the total alpha 1-AR mRNA in the human prostate. Furthermore, in situ hybridization localizes the alpha 1C AR mRNA predominantly to the stromal compartment. The identification of a predominant alpha 1-AR mRNA in human prostate identifies a potential need for subtype selective pharmaceutical agents. These agents could be very important clinically in the treatment of diseases such as BPH.

Humans↗
PubMed1993

Characterization of alpha 2-adrenergic receptor subtype-specific antibodies.

Subtypes of alpha 2-adrenergic receptors have been defined pharmacologically in a variety of mammalian tissues. The alpha 2A, alpha 2B, alpha 2C, and most recently alpha 2D subtypes have been characterized by their affinities for selective receptor antagonists and agonists. The genes that may encode the alpha 2A, alpha 2B, and alpha 2C subtypes have been identified in human and rat. In human these genes are termed alpha 2-C10, alpha 2-C2, and alpha 2-C4, respectively, based on their chromosomal localization, whereas three genes, designated RG20 alpha 2, RNG alpha 2, and RG10 alpha 2, are thought to be the corresponding rat homologues. These assignments were based on the pharmacology of the cloned receptor genes expressed in transfected cells and on the detection of homologous mRNAs by Northern blot analyses in cell lines or tissues with pharmacologically defined alpha 2-adrenergic receptors. However, the subtype assignment of cloned genes has not been fully resolved by these means. To help clarify the subtype assignment, we have raised antibodies against sequences from the divergent third intracellular loop of the human and rat alpha 2-adrenergic receptors. These antibodies were found to be subtype specific in immunoprecipitating either the cloned receptors expressed by DNA transfection or the pharmacologically defined receptors prepared from various tissues. Our immunological data corroborate the assignments of alpha 2-C2/RNG alpha 2 as encoding the alpha 2B subtype in NG108-15 cells and rat neonatal lung and of alpha 2-C4/RG10 alpha 2 as encoding the alpha 2C subtype in opossum kidney cells. Furthermore, antibodies against alpha 2-C10 and RG20 alpha 2 but not alpha 2-C2/RNG alpha 2 or alpha 2-C4/RG10 alpha 2 were both found to recognize alpha 2-adrenergic receptors expressed in rat submaxillary glands and in bovine pineal gland, two tissues with alpha 2D pharmacology. Because three genes were identified in the rat and human genome, these data suggest that the pharmacologically defined "alpha 2D receptor" is genetically of the alpha 2A subtype.

Adrenergic alpha-Agonists↗