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R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 145 records · Page 8Linked to original sources
PubMed1996

Myocardial beta-adrenergic receptor signaling in vivo: insights from transgenic mice.

Heart failure is a problem of increasing importance in cardiovascular medicine. An important characteristic of heart failure is reduced agonist-stimulated adenylyl cyclase activity (receptor desensitization) due to both diminished receptor number (receptor downregulation) and impaired receptor function (receptor uncoupling). These changes in the section-adrenergic receptor (section-AR) system may in part account for some of the abnormalities of contractile function in this disease. Myocardial contraction is closely regulated by G protein coupled beta-adrenergic receptors through the action of the second messenger cAMP. The beta-adrenergic receptors themselves are regulated by a set of specific kinases, termed the G-protein-coupled receptor kinases. The study of this complex system in vivo has recently been advanced by the development of transgenic and gene targeted ("knockout") mouse models. Combining transgenic technology with sophisticated physiological measurements of cardiac hemodynamics is an extremely powerful strategy to study the regulation of myocardial contractility in the normal and failing heart.

Animals↗
PubMed1996

Cloning, characterization, and chromosomal localization of rec1.3, a member of the G-protein-coupled receptor family highly expressed in brain.

During a project to identify G-protein-coupled receptors (GPCR) expressed within taste buds, we have isolated a novel receptor-like sequence. The full length sequence of this receptor (rec1.3) has been obtained in both cow and mouse. Rec1.3 bears little sequence similarity to any GPCR whose ligand is known: the closest identity (33%) is to the orphan receptor edg-1. In cow, rec1.3 is expressed most prominently in the brain, with moderate expression in testis and tongue; in the mouse the expression is more widespread. No specific binding for a range of ligands was detected when the mouse coding sequence was expressed in eukaryotic cells. In situ hybridization showed that rec1.3 is widely expressed throughout the mouse brain and is highly expressed in localized regions of the hindbrain, midbrain and hypothalamus. The rec1.3 gene was localized to the centromeric region of chromosome 4 in mouse, a region associated with neonatal seizures.

Amino Acid Sequence↗
PubMed1996

G protein-coupled receptor kinase mediates desensitization of norepinephrine-induced Ca2+ channel inhibition.

G protein-coupled receptors are essential signaling molecules at sites of synaptic transmission. Here, we explore the mechanisms responsible for the use-dependent termination of metabotropic receptor signaling in embryonic sensory neurons. We report that the inhibition of voltage-dependent Ca2+ channels mediated by alpha2-adrenergic receptors desensitizes slowly with prolonged exposure to the transmitter and that the desensitization is mediated by a G protein-coupled receptor kinase (GRK). Intracellular introduction of recombinant, purified kinases or synthetic blocking peptides into individual neurons demonstrates the specific involvement of a GRK3-like protein. These results suggest that GRK-mediated termination of receptor-G protein coupling is likely to regulate synaptic strength and, as such, may provide one effective mechanism for depression of synaptic transmission.

Animals↗
PubMed1996

Desensitization of G protein-coupled receptors.

Waning responsiveness to continuous or repeated stimulation constitutes the phenomenon of desensitization, which pervades biological systems. Over the last several years, molecular mechanisms for desensitization of cellular signaling through G protein-coupled receptors have been delineated, particularly at the level of the receptors themselves. This review focuses on those aspects of G protein-coupled receptor desensitization which occur within minutes of agonist exposure. Agonist-dependent desensitization of these receptors can reduce their signaling responsiveness to maximum stimulation by up to 70-80%; indeed, in some receptor systems, the process of receptor desensitization appears to effect the termination of the cellular signaling response. Agonist-induced desensitization involves phosphorylation of G protein-coupled receptors by two currently recognized classes of serine/threonine protein kinases. Second messenger-dependent kinases, phosphorylating a variety of proteins, mediate a generalized cellular hyporesponsiveness termed heterologous desensitization. G protein-coupled receptor kinases (GRKs) phosphorylate specifically agonist-occupied, or activated, receptors, and thereby initiate receptor-specific, or homologous, desensitization. GRK-mediated receptor phosphorylation facilitates the binding of an inhibitory arrestin protein to the phosphorylated receptor, an event which substantially impairs receptor signaling. The GRK family comprises six, and the arrestin family comprises four known members. Each GRK demonstrates the ability to phosphorylate a limited number of model receptor substrates, but the widespread expression of several GRKs and the two somatic arrestins suggest that GRK-initiated homologous desensitization is of general importance to a wide range of G protein-coupled receptors. Exploration of the substrate specificity for GRKs and the ligand specificity for arrestins remains in its early stages. Currently, GRKs can most readily be differentiated by the mechanisms each employs to associate with the plasma membrane. Accumulating data from human disease states and transgenic mice attest to the physiologic significance of GRK-initiated receptor desensitization.

Animals↗
PubMed1996

Production of adrenergic receptors in yeast.

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.

Adrenergic alpha-Antagonists↗
PubMed1995

G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.

The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Receptor-tyrosine-kinase- and G beta gamma-mediated MAP kinase activation by a common signalling pathway.

Mitogen-activated protein (MAP) kinases mediate the phosphorylation and activation of nuclear transcription factors that regulate cell growth. MAP kinase activation may result from stimulation of either tyrosine-kinase (RTK) receptors, which possess intrinsic tyrosine kinase activity, or G-protein-coupled receptors (GPCR). RTK-mediated mitogenic signalling involves a series of SH2- and SH3-dependent protein-protein interactions between tyrosine-phosphorylated receptor, Shc, Grb2 and Sos, resulting in Ras-dependent MAP kinase activation. The beta gamma subunits of heterotrimeric G proteins (G beta gamma) also mediate Ras-dependent MAP kinase activation by an as-yet unknown mechanism. Here we demonstrate that activation of MAP kinase by Gi-coupled receptors is preceded by the G beta gamma-mediated tyrosine phosphorylation of Shc, leading to an increased functional association between Shc, Grb2 and Sos. Moreover, disruption of the Shc-Grb2-Sos complex blocks G beta gamma-mediated MAP kinase activation, indicating that G beta gamma does not mediate MAP kinase activation by a direct interaction with Sos. These results indicate that G beta gamma-mediated MAP kinase activation is initiated by a tyrosine phosphorylation event and proceeds by a pathway common to both GPCRs and RTKs.

Adaptor Proteins, Signal Transducing↗
PubMed1995

The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.

Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.

Animals↗
PubMed1995

Phosphorylation and desensitization of the human beta 1-adrenergic receptor. Involvement of G protein-coupled receptor kinases and cAMP-dependent protein kinase.

Persistent stimulation of the beta 1-adrenergic receptor (beta 1AR) engenders, within minutes, diminished responsiveness of the beta 1 AR/adenylyl cyclase signal transduction system. This desensitization remains incompletely defined mechanistically, however. We therefore tested the hypothesis that agonist-induced desensitization of the beta 1AR (like that of the related beta 2AR) involves phosphorylation of the receptor itself, by cAMP-dependent protein kinase (PKA) and the beta-adrenergic receptor kinase (beta ARK1) or other G protein-coupled receptor kinases (GRKs). Both Chinese hamster fibroblast and 293 cells demonstrate receptor-specific desensitization of the beta 1 AR within 3-5 min. Both cell types also express beta ARK1 and the associated inhibitory proteins beta-arrestin-1 and beta-arrestin-2, as assessed by immunoblotting. Agonist-induced beta 1AR desensitization in 293 cells correlates with a 2 +/- 0.3-fold increase in phosphorylation of the beta 1AR, determined by immunoprecipitation of the beta 1AR from cells metabolically labeled with 32P(i). This agonist-induced beta 1AR phosphorylation derives approximately equally from PKA and GRK activity, as judged by intact cell studies with kinase inhibitors or dominant negative beta ARK1 (K220R) mutant overexpression. Desensitization, likewise, is reduced by only approximately 50% when PKA is inhibited in the intact cells. Overexpression of rhodopsin kinase, beta ARK1, beta ARK2, or GRK5 significantly increases agonist-induced beta 1AR phosphorylation and concomitantly decreases agonist-stimulated cellular cAMP production (p < 0.05). Furthermore, purified beta ARK1, beta ARK2, and GRK5 all demonstrate agonist-dependent phosphorylation of the beta 1AR. Consistent with a GRK mechanism, receptor-specific desensitization of the beta 1AR was enhanced by overexpression of beta-arrestin-1 and -2 in transfected 293 cells. We conclude that rapid agonist-induced desensitization of the beta 1AR involves phosphorylation of the receptor by both PKA and at least beta ARK1 in intact cells. Like the beta 2AR, the beta 1AR appears to bind either beta-arrestin-1 or beta-arrestin-2 and to react with rhodopsin kinase, beta ARK1, beta ARK2, and GRK5.

Amino Acid Sequence↗
PubMed1995

Ca(2+)-dependent interaction of recoverin with rhodopsin kinase.

Recoverin (Rv) is a myristoylated Ca(2+)-binding protein present primarily in bovine photoreceptors. It represents a newly identified family of neuronal specific Ca(2+)-binding proteins that includes neurocalcin, hippocalcin, and guanylyl cyclase-activating protein. To investigate the function of Rv in photoreceptors, we identified proteins that bind immobilized Rv in a Ca(2+)-dependent manner. Rhodopsin kinase (RK), interphotoreceptor retinoid-binding protein, and tubulin interact with Rv in the presence of Ca2+. The importance of the Rv/RK interaction was further characterized. RK, purified using immobilized Rv as an affinity matrix, catalyzed the light-dependent and Ca(2+)-independent incorporation of phosphates into rhodopsin when reconstituted with urea-stripped rod outer segment membranes. When only a small fraction (0.04%) of rhodopsin was photolyzed, as many as 700 phosphates were incorporated per photolyzed rhodopsin, a phenomenon known as "high gain" phosphorylation. When recoverin was added, the activity of RK became sensitive to free Ca2+, with EC50 = 3 microM. The N-terminal myristoyl residue of Rv enhances the inhibitory effect of Rv and introduces cooperativity to the Ca(2+)-dependent inhibition of rhodopsin phosphorylation. Rv neither interacts with other members of the G-protein-coupled receptor kinase family such as beta-adrenergic receptor kinase 1 nor inhibits beta-adrenergic receptor kinase 1 activity. The specific and Ca(2+)-dependent Rv/RK interaction is necessary for the inhibitory effect of Rv on rhodopsin phosphorylation and may play an important role in photoreceptor light adaptation.

Amino Acid Sequence↗
PubMed1995

Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation.

Receptors that couple to the heterotrimeric G proteins, Gi or Gq, can stimulate phosphoinositide (PI) hydrolysis and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC), which can stimulate increased MAPK activity. However, the relationship between PI hydrolysis and MAPK activation in Gi and Gq signaling has not been clearly defined and is the subject of this study. The effects of several signaling inhibitors are assessed including expression of a peptide derived from the carboxyl terminus of the beta adrenergic receptor kinase 1 (beta ARKct), which specifically blocks signaling mediated by the beta gamma subunits of G proteins (G beta gamma), expression of dominant negative mutants of p21ras (RasN17) and p74raf-1 (N delta Raf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC. The Gi-coupled alpha 2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of beta ARKct, RasN17, or N delta Raf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the Gq-coupled alpha 1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of beta ARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of N delta Raf or by PKC depletion. These data demonstrate that Gi- and Gq-coupled receptors stimulate MAPK activation via distinct signaling pathways. G beta gamma is responsible for mediating Gi-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, G alpha mediates Gq-coupled receptor-stimulated MAPK activation using a p21ras-independent mechanism employing PKC and p74raf. To define the role of G beta gamma in Gi-coupled receptor-mediated PI hydrolysis and MAPK activation, direct stimulation with G beta gamma was used. Expression of G beta gamma resulted in MAPK activation that was sensitive to inhibition by expression of beta ARKct, RasN17, or N delta Raf or by PTK inhibitors, but insensitive to PKC depletion. By comparison, G beta gamma-mediated PI hydrolysis was not affected by beta ARKct, RasN17, or N delta Raf expression or by PTK inhibitors. Together, these results demonstrate that G beta gamma mediates MAPK activation and PI hydrolysis via independent signaling pathways.

Animals↗
PubMed1995

G beta gamma subunits mediate mitogen-activated protein kinase activation by the tyrosine kinase insulin-like growth factor 1 receptor.

The receptors for insulin-like growth factor 1 (IGF1) and insulin are related heterotetrameric proteins which, like the epidermal growth factor (EGF) receptor, possess intrinsic ligand-stimulated tyrosine protein kinase activity. In Rat 1 fibroblasts, stimulation of mitogen-activated protein (MAP) kinase via the IGF1 receptor and the Gi-coupled receptor for lysophosphatidic acid (LPA), but not via the EGF receptor, is sensitive both to pertussis toxin treatment and to cellular expression of a specific G beta gamma subunit-binding peptide. The IGF1, LPA, and EGF receptor-mediated signals are all sensitive to inhibitors of tyrosine protein kinases, require p21ras activation, and are independent of protein kinase C. These data suggest that some tyrosine kinase growth factor receptors (e.g. IGF1 receptor) and classical G protein-coupled receptors (e.g. LPA receptor) employ a similar mechanism for mitogenic signaling that involves both tyrosine phosphorylation and G beta gamma subunits derived from pertussis toxin-sensitive G proteins.

Animals↗
PubMed1995

Mutational analysis of the pleckstrin homology domain of the beta-adrenergic receptor kinase. Differential effects on G beta gamma and phosphatidylinositol 4,5-bisphosphate binding.

The beta gamma subunits of heterotrimeric G proteins (G beta gamma) play a variety of roles in cellular signaling, one of which is membrane targeting of the beta-adrenergic receptor kinase (beta ARK). This is accomplished via a physical interaction of G beta gamma and a domain within the carboxyl terminus of beta ARK which overlaps with a pleckstrin homology (PH) domain. The PH domain of beta ARK not only binds G beta gamma but also interacts with phosphatidylinositol 4,5-bisphosphate (PIP2). Based on previous mapping of the G beta gamma binding region of beta ARK, and conserved residues within the PH domain, we have constructed a series of mutants in the carboxyl terminus of beta ARK in order to determine important residues involved in G beta gamma and PIP2 binding. To examine the effects of mutations on G beta gamma binding, we employed three different methodologies: direct G beta gamma binding to GST fusion proteins; the ability of GST fusion proteins to inhibit G beta gamma-mediated beta ARK translocation to rhodopsin-enriched rod outer segments; and the ability of mutant peptides expressed in cells to inhibit G beta gamma-mediated inositol phosphate accumulation. Direct PIP2 binding was also assessed on mutant GST fusion proteins. Ala residue insertion following Trp643 completely abolished the ability of beta ARK to bind G beta gamma, suggesting that a proper alpha-helical conformation is necessary for the G beta gamma.beta ARK interaction. In contrast, this insertional mutation had no effect on PIP2 binding. Both G beta gamma binding and PIP2 binding were abolished following Ala replacement of Trp643, suggesting that this conserved residue within the last subdomain of the PH domain is crucial for both interactions. Other mutations also produced differential effects on the physical interactions of the beta ARK carboxyl terminus with G beta gamma and PIP2. These results suggest that the last PH subdomain and its neighboring sequences within the carboxyl terminus of beta ARK, including Trp643, Leu647, and residues Lys663-Arg669, are critical for G beta gamma binding while Trp643 and residues Asp635-Glu639 are important for the PH domain to form the correct structure for binding to PIP2.

Amino Acid Sequence↗
PubMed1995

Effect of cellular expression of pleckstrin homology domains on Gi-coupled receptor signaling.

Pleckstrin homology (PH) domains are 90-110 amino acid regions of protein sequence homology that are found in a variety of proteins involved in signal transduction and growth control. We have previously reported that the PH domains of several proteins, including beta ARK1, PLC gamma, IRS-1, Ras-GRF, and Ras-GAP, expressed as glutathione S-transferase fusion proteins, can reversibly bind purified bovine brain G beta gamma subunits in vitro with varying affinity. To determine whether PH domain peptides would behave as antagonists of G beta gamma subunit-mediated signal transduction in intact cells, plasmid minigene constructs encoding these PH domains were prepared, which permit transient cellular expression of the peptides. Pertussis toxin-sensitive, G beta gamma subunit-mediated inositol phosphate (IP) production was significantly inhibited in COS-7 cells transiently coexpressing the alpha 2-C10 adrenergic receptor (AR) and each of the PH domain peptides. Pertussis toxin-insensitive, Gq alpha subunit-mediated IP production via coexpressed M1 muscarinic acetylcholine receptor (M1 AChR) was attenuated only by the PLC gamma PH domain peptide, suggesting that the inhibitory effect of most of the PH domain peptides was G beta gamma subunit-specific. Stimulation of the mitogen-activated protein (MAP) kinase pathway by Gi-coupled receptors in COS-7 cells has been reported to require activation of p21ras and to be independent of protein kinase C. Since several proteins involved in activation contain PH domains, the effect of PH domain peptide expression on alpha 2-C10 AR-mediated p21ras-GTP exchange and MAP kinase activation as well as direct G beta gamma subunit-mediated activation of MAP kinase was determined. In each assay, coexpression of the PH domain peptides resulted in significant inhibition. Increasing G beta gamma subunit expression surmounted PH domain peptide-mediated inhibition of MAP kinase activation. These data suggest that the PH domain peptides behave as specific antagonists of G beta gamma-mediated signaling in intact cells and that interactions between PH domains and G beta gamma subunits or structurally related proteins may play a role in the activation of mitogenic signaling pathways by G protein-coupled receptors.

Animals↗
PubMed1995

Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a beta ARK inhibitor.

Transgenic mice were created with cardiac-specific overexpression of the beta-adrenergic receptor kinase-1 (beta ARK1) or a beta ARK inhibitor. Animals overexpressing beta ARK1 demonstrated attenuation of isoproterenol-stimulated left ventricular contractility in vivo, dampening of myocardial adenylyl cyclase activity, and reduced functional coupling of beta-adrenergic receptors. Conversely, mice expressing the beta ARK inhibitor displayed enhanced cardiac contractility in vivo with or without isoproterenol. These animals demonstrate the important role of beta ARK in modulating in vivo myocardial function. Because increased amounts of beta ARK1 and diminished cardiac beta-adrenergic responsiveness characterize heart failure, these animals may provide experimental models to study the role of beta ARK in heart disease.

Adenylyl Cyclases↗