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R T Premont

Publications and source records attributed to R T Premont.

At least 37 records · Page 2Linked to original sources
PubMed1998

A role for receptor kinases in the regulation of class II G protein-coupled receptors. Phosphorylation and desensitization of the secretin receptor.

The secretin receptor is a member of a structurally distinct class of G protein-coupled receptors designated as Class II. The molecular mechanisms of secretin receptor signal termination are unknown. Using transiently transfected HEK 293 cells expressing the secretin receptor, we investigated its mechanisms of desensitization. Binding of [125I]-secretin to plasma membranes of receptor-expressing cells was specific, with a Kd of 2 nM. Secretin evoked an increase in cellular cAMP with an EC50 of 0.4 nM. The response was maximal by 20 min and desensitized rapidly and completely. Immunoprecipitation of a functional, N-terminal epitope-tagged secretin receptor was used to demonstrate agonist-dependent receptor phosphorylation, with an EC50 of 14 nM. Pretreatment with protein kinase A or C inhibitors failed to alter secretin-stimulated cAMP accumulation. G protein-coupled receptor kinases (GRKs) are known to be involved in the desensitization of Class I G protein-coupled receptors; therefore, the effect of cotransfection of GRKs on secretin-stimulated cAMP signaling and phosphorylation was evaluated. GRKs 2 and 5 were the most potent at augmenting desensitization, causing a 40% reduction in the maximal cAMP response to secretin. GRK 5 also caused a shift in the EC50 to the right (p < 0.05). GRK 4 and GRK 6 did not alter dose-dependent signaling, and GRK 3 was intermediate in effect. Receptor phosphorylation correlated with desensitization for each GRK studied, whereas second messenger-dependent kinase phosphorylation appeared to be less important in secretin receptor signal termination. We demonstrate agonist-dependent secretin receptor phosphorylation coincident with profound receptor desensitization of the signaling function in HEK 293 cells, suggesting a role for receptor phosphorylation in this paradigm. Although GRK activity appears important in secretin receptor desensitization in HEK 293 cells, protein kinases A and C appear to play only a minor role. These results demonstrate that the GRK-arrestin system regulates Class II G protein-coupled receptors.

Cell Line↗
PubMed1996

Phosphatidylinositol 4,5-bisphosphate (PIP2)-enhanced G protein-coupled receptor kinase (GRK) activity. Location, structure, and regulation of the PIP2 binding site distinguishes the GRK subfamilies.

The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors and play an important role in mediating receptor desensitization. The localization of these enzymes to their membrane incorporated substrates is required for their efficient function and appears to be a highly regulated process. In this study we demonstrate that phosphatidylinositol 4, 5-bisphosphate (PIP2) enhances GRK5-mediated beta-adrenergic receptor (betaAR) phosphorylation by directly interacting with this enzyme and facilitating its membrane association. GRK5-mediated phosphorylation of a soluble peptide substrate is unaffected by PIP2, suggesting that the PIP2-enhanced receptor kinase activity arises as a consequence of this membrane localization. The lipid binding site of GRK5 exhibits a high degree of specificity and appears to reside in the amino terminus of this enzyme. Mutation of six basic residues at positions 22, 23, 24, 26, 28, and 29 of GRK5 ablates the ability of this kinase to bind PIP2. This region of the GRK5, which has a similar distribution of basic amino acids to the PIP2 binding site of gelsolin, is highly conserved between members of the GRK4 subfamily (GRK4, GRK5, and GRK6). Indeed, all the members of the GRK4 subfamily exhibit PIP2-dependent receptor kinase activity. We have shown previously that the membrane association of betaARK (beta-adrenergic receptor kinase) (GRK2) is mediated, in vitro, by the simultaneous binding of PIP2 and the betagamma subunits of heterotrimeric G proteins to the carboxyl-terminal pleckstrin homology domain of this enzyme (Pitcher, J. A., Touhara, K., Payne, E. S., and Lefkowitz, R. J. (1995) J. Biol. Chem. 270, 11707-11710). Thus, five members of the GRK family bind PIP2, betaARK (GRK2), betaARK2 (GRK3), GRK4, GRK5, and GRK6. However, the structure, location, and regulation of the PIP2 binding site distinguishes the betaARK (GRK2 and GRK3) and GRK4 (GRK4, GRK5, and GRK6) subfamilies.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of a widely expressed form of adenylyl cyclase.

A novel mammalian adenylyl cyclase was identified by reverse transcription-polymerase chain reaction amplification using degenerate primers based on a conserved region of previously described adenylyl cyclases (Premont, R. T. (1994) Methods Enzymol. 238, 116-127). The full-length cDNA sequence obtained from mouse brain predicts a 1353-amino acid protein possessing a 12-membrane span topology, and containing two regions of high similarity with the catalytic domains of adenylyl cyclases. Comparison of this novel adenylyl cyclase with the eight previously described mammalian enzymes indicates that this type 9 adenylyl cyclase sequence is the most divergent, defining a sixth distinct subclass of mammalian adenylyl cyclases. The AC9 gene has been localized to human chromosome band 16p13.3-13.2. The 8.5-kb mRNA encoding the type 9 adenylyl cyclase is widely distributed, being readily detected in all tissues tested, and is found at very high levels in skeletal muscle and brain. AC9 mRNA is found throughout rat brain but is particularly abundant in hippocampus, cerebellum, and neocortex. An antiserum directed against the carboxyl terminus of the type 9 adenylyl cyclase detects native and expressed recombinant AC9 protein in tissue and cell membranes. Levels of the AC9 protein are highest in mouse brain membranes. Characterization of expressed recombinant AC9 reveals that the protein is a functional adenylyl cyclase that is stimulated by Mg2+, forskolin, and mutationally activated Gsalpha. AC9 activity is not affected by Ca2+/calmodulin or by G protein betagamma-subunits. Thus AC9 represents a functional G protein-regulated adenylyl cyclase found in brain and in most somatic tissues.

Adenylyl Cyclases↗
PubMed1996

Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration.

We recently reported that a beta2-adrenergic receptor (beta2AR) mutant, Y326A, defective in its ability to sequester in response to agonist stimulation was a poor substrate for G protein-coupled receptor kinase (GRK)-mediated phosphorylation; however, its ability to be phosphorylated and sequestered could be restored by overexpressing GRK2 [Ferguson et al. (1995) J. Biol. Chem. 270, 24782]. In the present report, we tested the ability of each of the known GRKs (GRK1-6) to phosphorylate and rescue the sequestration of the Y326A mutant in HEK-293 cells. We demonstrate that in addition to GRK2, GRK3-6 can phosphorylate the Y326A mutant and rescue its sequestration; however, GRK1 was totally ineffective in rescuing either the phosphorylation or the sequestration of the mutant receptor. We found that the agonist-dependent rescue of Y326A mutant phosphorylation by GRK2, -3, and -5 was associated with the agonist-dependent rescue of sequestration. In contrast, overexpression of GRK4 and -6 led mainly to agonist-independent phosphorylation of the Y326A mutant accompanied by increased basal receptor sequestration. Our results demonstrate that phosphorylation per se, but not the interaction with a specific GRK, is required to facilitate beta2AR sequestration.

Adrenergic beta-Antagonists↗
PubMed1996

Characterization of the G protein-coupled receptor kinase GRK4. Identification of four splice variants.

A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.

Alternative Splicing↗
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↗
PubMed1995

Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses.

Only three isoforms of adenylyl cyclase (EC 4.6.1.1) mRNAs (AC1, -2, and -5) are expressed at high levels in rat brain. AC1 occurs predominantly in hippocampus and cerebellum, AC5 is restricted to the basal ganglia, whereas AC2 is more widely expressed, but at much lower levels. The distribution and abundance of adenylyl cyclase protein were examined by immunohistochemistry with an antiserum that recognizes a peptide sequence shared by all known mammalian adenylyl cyclase isoforms. The immunoreactivity in striatum and hippocampus could be readily interpreted within the context of previous in situ hybridization studies. However, extending the information that could be gathered by comparisons with in situ hybridization analysis, it was apparent that staining was confined to the neuropil--corresponding to immunoreactive dendrites and axon terminals. Electron microscopy indicated a remarkably selective subcellular distribution of adenylyl cyclase protein. In the CA1 area of the hippocampus, the densest immunoreactivity was seen in postsynaptic densities in dendritic spine heads. Labeled presynaptic axon terminals were also observed, indicating the participation of adenylyl cyclase in the regulation of neurotransmitter release. The selective concentration of adenylyl cyclases at synaptic sites provides morphological data for understanding the pre- and postsynaptic roles of adenylyl cyclase in discrete neuronal circuits in rat brain. The apparent clustering of adenylyl cyclases, coupled with other data that suggest higher-order associations of regulatory elements including G proteins, N-methyl-D-aspartate receptors, and cAMP-dependent protein kinases, suggests not only that the primary structural information has been encoded to render the cAMP system responsive to the Ca(2+)-signaling system but also that higher-order strictures are in place to ensure that Ca2+ signals are economically delivered and propagated.

Adenylyl Cyclases↗
PubMed1995

Rhodopsin kinase autophosphorylation. Characterization of site-specific mutations.

Upon illumination, rhodopsin kinase (RK) phosphorylates the visual pigment rhodopsin, which is thought to partially terminate the biochemical events that follow photon absorption. RK enzymology was explored by mutagenesis of the residues Ser488, Thr489 (major autophosphorylation sites), and Lys491 (a distal residue). We found the following to be true. (i) Double mutations at residues Ser488 and Thr489 to Ala or Asp decrease autophosphorylation to substoichiometrical levels, while single mutations at either residue independently reduce autophosphorylation by half. (ii) Phosphorylation of residue Ser488 influences the affinity of RK for heparin-Sepharose only moderately, whereas Thr489 and Lys491 are important for this interaction. RK K491A does not phosphorylate acidic peptides, suggesting that this residue participates in substrate binding. (iii) Mutations in the autophosphorylation region affect the Km for ATP, suggesting that this region is involved in binding of ATP to the catalytic site. (iv) RK mutants S488A or S488D and RK S488A and T489A have an increased ability to phosphorylate Rho in the dark. (v) Mutations at the autophosphorylation region change the initial site of phosphorylation on photolyzed rhodopsin (Rho*), implying that this region may regulate selectivity of the site of phosphorylation.

Amino Acid Sequence↗
PubMed1995

Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines.

Immortalized brown adipocyte cell lines derived from a mouse hibernoma express all three beta-adrenergic receptor subtypes, including beta 3-adrenergic receptor (AR). In response to norepinephrine, cAMP production by plasma membranes from four clonal cell lines was stimulated to levels comparable with brown adipocytes isolated from interscapular brown adipose tissue (72.8-89.6 versus 97.8 pmol cAMP/min/mg of protein, respectively). All cell lines responded to the highly selective beta 3-adrenergic receptor agonist CL316,243 by stimulating adenylyl cyclase activity (3-10-fold over basal). beta 1-, beta 2-, and beta 3-adrenergic receptor mRNA was detected by Northern blotting and/or reverse transcriptase-polymerase chain reaction. Competition binding assays with the antagonists CGP20712A and 125I-cyanopindolol showed the proportions of beta 1AR and beta 2AR in immortalized cells to be similar to brown adipocytes from tissue (cells: 35% beta 1AR, 65% beta 2AR; brown adipocytes from tissue: beta 1AR 41%, 59% beta 2AR). Expression of brown fat-specific mitochondrial uncoupling protein (Ucp) was stimulated by beta-adrenergic agonists in two of the four cell lines. The ability of individual beta AR subtypes to regulate Ucp expression was examined with combinations of selective beta-adrenergic agonists and antagonists. Expression of Ucp could be induced by any of the beta-adrenergic receptor subtypes. However, the greatest response was obtained by stimulating all three beta-adrenergic receptor subtypes simultaneously (100 microM isoproterenol). Incubation of membranes from cultured cells or brown adipocytes from tissue with CL316,243 at an optimal concentration (5 microM) did not prevent norepinephrine from further stimulating adenylyl cyclase activity, suggesting that the combined activation of beta 1AR/beta 2AR, plus beta 3AR, together produced an additive cAMP response. Multiple forms of adenylyl cyclase were identified in brown and white adipocyte cell lines and tissues. Northern blot analysis detected adenylyl cyclase types 5, 6, and 10. Screening of reverse transcriptase-PCR products by DNA sequencing confirmed the identities of these forms and lower levels of additional isoforms, raising the possibility that beta-adrenergic receptor subtypes in adipocytes couple to distinct adenylyl cyclases. Because these cell lines display functional and phenotypic similarities to interscapular brown adipocytes, they will be a useful model to study the regulation of beta-adrenergic receptor expression and function, and the control of Ucp expression and activity.

Adenylyl Cyclases↗
PubMed1995

Protein kinases that phosphorylate activated G protein-coupled receptors.

G protein-coupled receptor kinases (GRKs) are a family of serine/threonine protein kinases that specifically recognize agonist-occupied, activated G protein-coupled receptor proteins as substrates. Phosphorylation of an activated receptor by a GRK terminates signaling by that receptor, by initiating the uncoupling of the receptor from heterotrimeric G proteins. Six distinct mammalian GRKs are known, which differ in tissue distribution and in regulatory properties. The intracellular localization of GRKs to membrane-bound receptor substrates is the most important known regulatory feature of these enzymes. Rhodopsin kinase (GRK1) requires a post-translationally added farnesyl isoprenoid to bind to light-activated rhodopsin. The beta-adrenergic receptor kinases (GRK2 and GRK3) associate with heterotrimeric G protein beta gamma-subunits, released upon receptor activation of G proteins, for membrane anchorage. The recently-described GRKs 4, 5, and 6 comprise a distinct subgroup of GRKs. These kinases utilize distinct mechanisms for membrane localization, which are just beginning to be defined. All GRKs appear to play the same general cellular role of desensitizing activated G protein-coupled receptors, but utilize distinctly individual means to the same end.

Amino Acid Sequence↗
PubMed1994

Palmitoylation of G protein-coupled receptor kinase, GRK6. Lipid modification diversity in the GRK family.

GRK6, a 66-kDa serine/threonine protein kinase, is a recently identified member of the G protein-coupled receptor kinase (GRK) family. GRKs are involved in the phosphorylation of seven-transmembrane receptors, a process mediating desensitization of signal transduction. An important feature of these enzymes is their membrane-associated nature, which for some members is stimulus-dependent. The structural basis for this membrane association previously has been shown in different members of the GRK family to include isoprenylation, G protein beta gamma-binding domains, and basic regions to provide electrostatic interactions with phospholipids. We provide evidence that another mechanism includes fatty acid acylation. GRK6, but not other GRKs tested, incorporated tritium after incubation with [3H]palmitate in Sf9 and in COS-7 cells overexpressing the kinase. The incorporated radioactivity was released from the protein by neutral hydroxylamine, indicating the presence of a thioester bond, and was confirmed as palmitic acid by high performance liquid chromatography analysis. Site-directed mutagenesis defined the region of palmitate attachment as a cluster of 3 cysteines (Cys561, Cys562, and Cys565) in the carboxyl-terminal domain of the kinase, consistent with the location of the membrane targeting domains of GRKs 1, 2, 3, and 5. Palmitoylation of GRK6 appears essential for membrane association, since palmitoylated kinase was found only in the membrane fraction. This lipid modification provides a structural basis for potential regulation of the subcellular distribution of GRK6 through acylation/deacylation cycles.

Acylation↗
PubMed1994

Identification, purification, and characterization of GRK5, a member of the family of G protein-coupled receptor kinases.

A novel member of the family of G protein-coupled receptor kinases (GRKs), named GRK5, has been cloned from bovine taste epithelium. The cDNA sequence predicts a 590-amino acid protein with high overall similarity to rhodopsin kinase. GRK5 mRNA is found most abundantly in lung, heart, retina, and lingual epithelium, but is expressed very little in brain, liver, kidney, or testis. GRK5 expressed in Sf9 cells was purified to apparent homogeneity. GRK5 major autophosphorylation sites were mapped to Ser484 and Thr485. Purified GRK5 phosphorylates rhodopsin in a light-dependent manner and beta 2-adrenergic receptor in an agonist-dependent manner and phosphorylates the C-terminal tail regions of both receptor proteins. GRK5 possesses neither a CAAX motif specifying protein prenylation like rhodopsin kinase nor similarity to the G protein beta gamma-subunit binding domain of beta-adrenergic receptor kinases. GRK5 phosphorylation of rhodopsin or beta 2-adrenergic receptor is not stimulated by G protein beta gamma-subunits. The GRK5 protein does not undergo agonist-dependent translocation from cytosol to membranes as do beta-adrenergic receptor kinase and rhodopsin kinase, but rather appears to associate with membranes constitutively. GRK5 thus appears functionally similar to other characterized GRKs, but has distinct regulatory properties which may be important for its cellular function.

Amino Acid Sequence↗
PubMed1993

Receptor-evoked Cl- current in Xenopus oocytes is mediated through a beta-type phospholipase C. Cloning of a new form of the enzyme.

Xenopus oocytes exhibit a receptor-evoked Cl- current that is mediated through the activation of phospholipase C (PLC) and release of intracellular Ca2+. The identity of PLC(s) mediating this effect is unknown. We have cloned cDNAs encoding a new form of PLC-beta from a Xenopus oocyte cDNA library. The Xenopus PLC-beta has substantial (33-64%) homology with mammalian beta 1, beta 2, beta 3, and beta 4 phospholipase C and is closest to PLC-beta 3, with 64% identity and 80% similarity. Injection of antisense oligonucleotides to a specific region of Xenopus PLC-beta results in degradation of its mRNA and significantly reduces Cl- currents evoked by both endogenous angiotensin receptors and expressed mammalian alpha 1b-adrenergic receptors and M1-muscarinic receptors as compared to responses in sense oligonucleotide-injected oocytes. Inhibition of the M1-muscarinic response by antisense oligonucleotides was nonadditive with pertussis toxin inhibition. PLC antisense oligonucleotide-injected oocytes show Cl- current responses to IP3 that are indistinguishable from sense oligonucleotide-injected oocytes. Since the receptor responses are pertussis toxin-sensitive, we conclude that we have isolated a new form of PLC-beta involved in the pertussis toxin-sensitive receptor stimulation of the Ca2+ activated Cl- current in Xenopus oocytes.

Amino Acid Sequence↗
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↗