Showing papers by "Robert J. Lefkowitz published in 1994"

Enhanced myocardial function in transgenic mice overexpressing the beta 2-adrenergic receptor

Abstract: Transgenic mice were created with cardiac-specific overexpression of the beta 2-adrenergic receptor. This resulted in increased basal myocardial adenylyl cyclase activity, enhanced atrial contractility, and increased left ventricular function in vivo; these parameters at baseline in the transgenic animals were equal to those observed in control animals maximally stimulated with isoproterenol. These results illustrate a useful approach for studying the effect of gene expression on cardiac contractility. Because chronic heart failure in humans is accompanied by a reduction in the number of myocardial beta-adrenergic receptors and in inotropic responsiveness, these results suggest a potential gene therapy approach to this disease state.

Activation of the cloned muscarinic potassium channel by G protein βγ subunits

Abstract: ACETYLCHOLINE released during parasv mpathetic stimulation of the vagal nerve slows the heart rate through the activation of muscarinic receptors and subsequent opening of an inwardly rectifying potassium channel1. The activation of these muscarinic potassium channels is mediated by a pertussis toxin-sensitive heterotrimeric GTP-binding protein (G protein)2,3. It has not been resolved whether exogenously applied Gα4,5 or Gβγ6,7, or both, activate the channel. Using a heterologous expression system, we have tested the ability of different G protein subunits to activate the cloned muscarinic potassium channel, GIRK18,9. We report here that coexpression of GIRK1 with Gβγ but not Gαβγ in Xenopus oocytes results in channel activity that persists in the absence of cytoplasmic GTP. This activity is reduced by fusion proteins of the (β-adrenergic receptor kinase and of recombinant Gαi–GDP, both of which are known to interact with Gβγ10,11. Moreover, application of recombinant Gβγ, but not Gαi–GTP-γS, activates GIRK1 channels. Thus Gβγ appears to be sufficient for the activation of GIRK1 muscarinic potassium channels.

Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras

Abstract: Stimulation of Gi-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21ras (Ras). Which G-protein subunit(s) (G alpha or the G beta gamma complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G beta gamma-binding domain, is a cellular G beta gamma antagonist capable of specifically distinguishing G alpha- and G beta gamma-mediated processes. Using this G beta gamma inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G beta gamma antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (alpha 2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G beta gamma antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (alpha 1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G beta gamma antagonist. These results identify G beta gamma as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.

Myocardial expression of a constitutively active alpha 1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy.

Abstract: Transgenic mice were generated by using the alpha-myosin heavy chain promoter coupled to the coding sequence of a constitutively active mutant alpha 1B-adrenergic receptor (AR). These transgenic animals demonstrated cardiac-specific expression of this alpha 1-AR with resultant activation of phospholipase C as shown by increased myocardial diacylglycerol content. A phenotype consistent with cardiac hypertrophy developed in adult transgenic mice with increased heart/body weight ratios, myocyte cross-sectional areas, and ventricular atrial natriuretic factor mRNA levels relative to nontransgenic controls. These transgenic animals may provide insight into the biochemical triggers that induce hypertrophy in cardiac disease and serve as a convenient experimental model for studies of this condition.

Negative antagonists promote an inactive conformation of the beta 2-adrenergic receptor.

Abstract: The beta 2-adrenergic receptor undergoes isomerization between an inactive conformation (R) and an active conformation (R*). The formation of the active conformation of the receptor molecule can be promoted by adrenergic agonists or by mutations in the third cytoplasmic domain that constitutively activate the receptor. Here we show that, of several beta-adrenergic receptor-blocking drugs tested, only two, ICI 118551 and betaxolol, inhibit the basal signaling activity of the beta 2-adrenergic receptor, thus acting as negative antagonists. We document the molecular properties of the more efficacious ICI 118551; (i) it shows higher affinity for the inactive form of the receptor and (ii) it inhibits the spontaneous formation of a beta-adrenergic receptor kinase substrate by the receptor. These properties are opposite those of adrenergic agonists, indicating that, in a fashion reciprocal to that of agonists, negative antagonists promote the formation of an inactive conformation of the receptor.

Localization of mRNA for three distinct alpha 1-adrenergic receptor subtypes in human tissues: implications for human alpha-adrenergic physiology.

Abstract: alpha 1-Adrenergic receptors (alpha 1ARs) are virtually ubiquitous in human tissues and mediate important physiological functions as diverse as smooth muscle contraction, glycogenolysis, and myocardial inotropy. At least three alpha 1AR subtypes (alpha 1A/D, alpha 1B, and alpha 1C) have been described using molecular and pharmacological techniques. The identification of species heterogeneity (rat versus rabbit) in alpha 1AR subtype distribution has made it imperative to determine the distribution of alpha 1AR subtypes in human tissues. Accordingly, RNA extracted from human tissues was analyzed using RNase protection assays to determine alpha 1AR subtype expression. Of the cloned alpha 1ARs, alpha 1CAR mRNA predominates in many human tissues (heart, liver, cerebellum, and cerebral cortex), in contrast to its restricted distribution in both rats and rabbits. alpha (1B)AR mRNA is present in highest concentrations in human spleen, kidney, and fetal brain. alpha 1A/DAR mRNA is present in highest concentrations in human aorta and cerebral cortex. Hence, alpha 1AR subtype mRNA distribution is tissue selective and differs from that reported for rats and rabbits. These results have potentially significant implications for understanding human adrenergic physiology and are important for the rational development of alpha 1AR subtype-selective drugs.

A constitutively active mutant beta 2-adrenergic receptor is constitutively desensitized and phosphorylated

Abstract: The beta 2-adrenergic receptor (beta 2AR) can be constitutively activated by mutations in the third intracellular loop. Whereas the wild-type receptor exists predominantly in an inactive conformation (R) in the absence of agonist, the mutant receptor appears to spontaneously adopt an active conformation (R*). We now demonstrate that not only is the mutant beta 2AR constitutively active, it is also constitutively desensitized and down-regulated. To assess whether the mutant receptor can constitutively engage a known element of the cellular desensitization machinery, the receptor was purified and reconstituted into phospholipid vesicles. These preparations retained the essential properties of the constitutively active mutant receptor: agonist-independent activity [to stimulate guanine nucleotide-binding protein (Gs)-GTPase] and agonist-specific increase in binding affinity. Moreover, the purified mutant receptor, in the absence of agonist, was phosphorylated by recombinant beta AR-specific kinase (beta ARK) in a fashion comparable to the agonist-occupied wild-type receptor. Thus, the conformation of the mutated receptor is equivalent to the active conformation (R*), which stimulates Gs protein and is identical to the beta ARK substrate.

Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase

Abstract: The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate. The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain. We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. Gi alpha-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain.

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

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.

INHIBITION OF beta ARK AND beta -ARRESTIN

Abstract: beta -adrenergic receptor kinase ( beta ARK) and beta -arrestin function in the homologous or agonist-activated desensitization of G-protein coupled receptors. Both beta ARK-2 and beta -arrestin-2 isoforms are highly enriched in and localized to the cilia and dendritic knobs of the olfactory receptor neurons where the initial events of olfactory signal transduction occur as well as being localized to spermatids. Administration of neutralizing antibodies to beta ARK-2 and beta -arrestin-2 enhances the response to odorants and attenuates desensitization.

INHIBITION OF βARK AND β-ARRESTIN

Abstract: β-adrenergic receptor kinase (βARK) and β-arrestin function in the homologous or agonist-activated desensitization of G-protein coupled receptors. Both βARK-2 and β-arrestin-2 isoforms are highly enriched in and localized to the cilia and dendritic knobs of the olfactory receptor neurons where the initial events of olfactory signal transduction occur as well as being localized to spermatids. Administration of neutralizing antibodies to βARK-2 and β-arrestin-2 enhances the response to odorants and attenuates desensitization.