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M M Kwatra

Publications and source records attributed to M M Kwatra.

33 records · Page 2Linked to original sources
PubMed1992

Role of beta gamma subunits of G proteins in targeting the beta-adrenergic receptor kinase to membrane-bound receptors.

The rate and extent of the agonist-dependent phosphorylation of beta 2-adrenergic receptors and rhodopsin by beta-adrenergic receptor kinase (beta ARK) are markedly enhanced on addition of G protein beta gamma subunits. With a model peptide substrate it was demonstrated that direct activation of the kinase could not account for this effect. G protein beta gamma subunits were shown to interact directly with the COOH-terminal region of beta ARK, and formation of this beta ARK-beta gamma complex resulted in receptor-facilitated membrane localization of the enzyme. The beta gamma subunits of transducin were less effective at both enhancing the rate of receptor phosphorylation and binding to the COOH-terminus of beta ARK, suggesting that the enzyme preferentially binds specific beta gamma complexes. The beta gamma-mediated membrane localization of beta ARK serves to intimately link receptor activation to beta ARK-mediated desensitization.

Amino Acid Sequence↗
PubMed1992

The ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.

To examine the role of the ligand binding domain of epidermal growth factor receptor in its dimerization, we studied the dimerization of a truncated form of the receptor that resembles v-erbB in that it lacks a ligand binding domain. Receptor dimerization was determined by sedimentation analysis on sucrose density gradients at different concentrations of Triton X-100. At high concentrations of Triton X-100 (0.2%), the truncated receptor occurred as a monomer and displayed low basal autophosphorylation. By contrast, at low concentrations of Triton X-100 (0.01%), it existed as a dimer and exhibited high basal autophosphorylation. The ability of the truncated receptor to dimerize indicates that the ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.

Autoradiography↗
PubMed1990

Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma.

We have investigated human gliomas that amplify and rearrange the epidermal growth factor receptor gene, with generation of an in-frame deletion mutation of 802 nucleotides in the external domain. This in-frame deletion mutation generates a local amino acid sequence at the fusion junction of what normally were distant polypeptide sequences in the intact epidermal growth factor receptor. This 14-amino acid peptide was chemically synthesized, coupled to keyhole limpet hemocyanin, and used as an immunogen in rabbits. The elicited antibody reacted specifically with the fusion peptide in ELISA. The anti-fusion junction peptide antibody was purified by passage of the antiserum over a peptide affinity column with acidic elution. The purified antibody selectively bound the glioma deletion mutant as compared to the intact epidermal growth factor receptor as assessed by immunocytochemistry, immunofluorescence, immunoprecipitation with gel electrophoresis, and binding experiments using radioiodinated antibody. These data indicate that it is feasible to generate site-specific anti-peptide antibodies that are highly selective for mutant proteins in human tumors. The anti-peptide antibody described here, and other mutation site-specific antibodies, should be ideal candidates for tumor immunoimaging and immunotherapy.

Amino Acid Sequence↗
PubMed1989

Phosphorylation of chick heart muscarinic cholinergic receptors by the beta-adrenergic receptor kinase.

Previous studies have demonstrated that muscarinic cholinergic receptors (mAChR) become markedly phosphorylated when intact cardiac cells are stimulated with a muscarinic agonist. This process appears to be related to the process of receptor desensitization. However, the mechanism of agonist-induced phosphorylation of mAChR is not known. In situ phosphorylation studies suggested that agonist-induced phosphorylation of mAChR may involve the participation of a receptor-specific kinase and/or require agonist occupancy. These observations regarding phosphorylation and desensitization of mAChR are similar to observations made for beta-adrenergic receptors. Recent studies have indicated that homologous desensitization of beta-adrenergic receptors may be due to the phosphorylation of these receptors by a novel protein kinase that only recognizes the agonist-occupied form of the receptors. As muscarinic receptors are structurally homologous to beta-adrenergic receptors, we have initiated studies to identify the protein kinase responsible for the phosphorylation of muscarinic receptors by determining whether the chick heart muscarinic receptor would serve as a substrate for the beta-adrenergic receptor kinase (beta-AR kinase). We report that the purified and reconstituted chick heart muscarinic receptor serves as an excellent substrate in vitro for the beta-AR kinase. Phosphorylation of mAChR receptors by the beta-AR kinase was only observed in the presence of a muscarinic receptor agonist and was prevented in the presence of antagonist. Both the extent of phosphorylation (3-4 mol of P/mol of receptor) and the phosphoamino acid composition of the mAChR after incubation in vitro with beta-AR kinase were similar to the characteristics of agonist-induced phosphorylation of mAChR in situ.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

The porcine heart M2 muscarinic receptor: agonist-induced phosphorylation and comparison of properties with the chick heart receptor.

Recently we showed that the chick heart muscarinic acetylcholine receptor is a phosphoprotein in intact cells and that treatment with agonists results in a striking increase in receptor phosphorylation [J. Biol. Chem. 261:12429-12432 (1986)]. Furthermore, we showed that the agonist-induced increase in the phosphorylation of chick heart muscarinic receptors correlates with receptor desensitization [J. Biol. Chem. 262:16314-16321 (1987)]. We have now extended studies of receptor phosphorylation to mammalian cardiac muscarinic receptors, in order to test the concept that phosphorylation is of general importance in the regulation of muscarinic receptor function. We have determined that, in intact porcine atria, M2 muscarinic receptors are phosphoproteins and that treatment with the agonist carbachol markedly increases receptor phosphorylation, to 4-6 mol of phosphate/mol of protein. Phosphorylation occurs on serine and threonine residues. Activation of either protein kinase C or cAMP-dependent protein kinase did not mimic the effect of agonists on receptor phosphorylation. These results are very similar to those seen with the chick heart muscarinic receptors. To determine whether the porcine and the chick cardiac muscarinic receptors represent similar or different proteins, we undertook detailed pharmacological studies and, in addition, prepared peptide maps of purified muscarinic receptors from chick heart and porcine atria. Our data show that there are marked differences in the pharmacological properties of the chick and the porcine cardiac muscarinic receptors. The peptide maps of the porcine and chick heart muscarinic receptors are also different, suggesting that muscarinic receptors in chick and porcine cardiac cells differ in their primary structure. Taken together, the data show that porcine and chick cardiac muscarinic receptors possess pharmacological and structural differences, but both receptors undergo agonist-mediated phosphorylation in intact cardiac cells. These data support the possibility that receptor phosphorylation may be of general importance in the regulation of muscarinic receptors.

Animals↗
PubMed1988

Characterization of cardiac A1 adenosine receptors by ligand binding and photoaffinity labeling.

[125I]N6-(p-aminobenzyl)adenosine and [125I]N6-(p-azidobenzyl)adenosine, which are potent agonists at A1 (Ri) adenosine receptors, have been used to characterize the adenosine receptor in membranes prepared from newborn chick heart. Scatchard analyses of [125I]N6-(p-aminobenzyl)adenosine binding to cardiac membranes revealed that the ligand bound to two affinity states of the receptor with Kd values of 0.7 and 9.9 nM. The corresponding maximum binding (Bmax) values were 25 and 86 fmol/mg of protein, respectively. In the presence of 0.1 mM 5'-guanylyl imidodiphosphate, a single affinity state was detected with a Kd of 9.4 nM and a Bmax of 96 fmol/mg of protein. Direct and indirect ligand binding studies with several adenosine receptor agonists and antagonists were used to compare the characteristics of the cardiac receptor with those of the A1 receptor in the cerebellum. The binding properties of the receptors in the two tissues were very similar although marked differences were observed in the binding kinetics of [125I]N6-(p-azidobenzyl)adenosine. Photo-affinity labeling experiments followed by sodium dodecyl sulfate-gel electrophoresis showed that the cardiac receptor had a apparent molecular weight of 37,600, which was slightly but significantly higher than that of the cerebellar receptor (35,500). The present results show that the cardiac receptor has ligand binding properties and a minimal subunit molecular weight similar to the more thoroughly studied A1 receptor in neural tissue.

Adenosine↗
PubMed1987

Correlation of agonist-induced phosphorylation of chick heart muscarinic receptors with receptor desensitization.

We have determined whether the process of agonist-mediated phosphorylation of the muscarinic receptor correlates with the process of muscarinic receptor desensitization in chick cardiac tissue. Exposure of ventricular slices to the agonist carbachol under conditions previously shown to lead to large increases in muscarinic receptor phosphorylation (Kwatra, M. M., and Hosey, M. M. (1986) J. Biol. Chem. 261, 12429-12432) resulted in decreased affinity of the muscarinic receptor for agonists. The agonist oxotremorine mimicked and the antagonist atropine prevented the effects of carbachol on receptor phosphorylation and agonist affinity. The time courses and concentration dependences for agonists to induce phosphorylation of the muscarinic receptor and decreases in agonist affinity were similar. Treatment of chick atria with acetylcholine under conditions which led to receptor phosphorylation resulted in decreased sensitivity of these preparations to the negative inotropic effect of carbachol. Taken together, the results support the concept that phosphorylation of cardiac muscarinic receptors may be related to the process of receptor desensitization. The mechanism by which agonists induce receptor phosphorylation was also investigated. The phosphorylated amino acids formed in response to agonists were serine and threonine. The protein kinase C activator phorbol myristate acetate had no effect on receptor phosphorylation or agonist affinity, nor did it prevent the effects of carbachol on either of these parameters. Receptor phosphorylation also was unaffected by the calmodulin antagonists W-7 and W-13, by elevation of cyclic nucleotides, and by agonists which activate other cardiac receptor systems. The results suggest that the phosphorylation of cardiac muscarinic receptors requires agonist occupancy of the receptor and/or may involve the participation of a selective protein kinase.

Animals↗
PubMed1987

N6-phenyladenosines: pronounced effect of phenyl substituents on affinity for A2 adenosine receptors.

A number of N6-phenyladenosines with various substitutions on the phenyl ring have been synthesized and tested for their affinities toward brain A1 and A2 adenosine receptors. Compounds with meta substituents, such as (m-hydroxy- and m-iodophenyl)adenosine, were found to have high A1 selectivity. Meta substitution caused a selective decrease in the affinity of these compounds for A2 receptors. The results suggest that, in contrast to what is commonly held, certain N6-substituents have pronounced effects on affinity for brain A2 adenosine receptors. Thus, brain A2 receptors may have a well-defined region that recognizes N6-substitutions.

Adenosine↗
PubMed1986

Phosphorylation of the cardiac muscarinic receptor in intact chick heart and its regulation by a muscarinic agonist.

We have tested the possibility that regulation of cardiac muscarinic receptor function may involve receptor phosphorylation. Chick heart muscarinic receptors were purified from relatively small amounts of tissue to near homogeneity using a three-step chromatographic procedure that utilized the affinity chromatography procedure of Haga and Haga (Haga, K., and Haga, T. (1983) J. Biol. Chem. 258, 13575-13579). The purified preparations contained a single major peptide which migrated on sodium dodecyl sulfate gels with an apparent Mr of 79,000. When receptors were purified from 32P-bathed hearts, a single major phosphopeptide eluted from the affinity column and comigrated on sodium dodecyl sulfate gels with the band of stained receptor. Treatment of hearts with the agonist carbachol led to marked increases (10-12-fold) in the phosphorylation of the receptor. The results show that the muscarinic receptor is a phosphoprotein in cardiac tissue and that treatment with a receptor agonist regulates its phosphorylation in the intact cell.

Animals↗
PubMed1985

Specific photoaffinity labelling of inhibitory adenosine receptors.

N6(L-phenylisopropyl)adenosine (L-PIA) and N6(3-iodo-4-azido benzyl)-adenosine (IAzBA) inhibit the adenylate cyclase activity in synaptic membranes of chick cerebellum via Ri adenosine receptors. [3H]L-PIA and [125I]AzBA bind to these membranes with Kd values of approximately 1 nM and Bmax values of approximately 1000 fmol/mg protein. Photolysis of [125I]AzBA bound to synaptic membranes results in the specific incorporation of radioactivity into a protein with Mr = 36,000. This photoincorporation is blocked by simultaneous exposure to L-PIA, theophylline, an adenosine receptor antagonist, or Gpp(NH)p, but not by cytosine, suggesting that the 36,000 dalton protein is the Ri adenosine receptor or a subunit of the receptor that contains the adenosine binding site.

Adenosine↗
PubMed1978

Acetylenics. 2. Synthesis and pharmacology of certain N,N-diakyl-3-phenylpropyn-2-amines. Some analogues with tryptamine-like behavioral effects in mice.

A number of N,N-dialkyl-3-phenylpropyn-2-amines 7 have been prepared and tested for their biological action. Certain analogues show tryptamine-like behavior effects in mice. The tryptamine-like activity of these compounds appears to be controlled by their lipophilicity. These compounds show only weak inhibition of rat liver monoamine oxidase. Although these compounds exhibit tryptamine-like action, experiments seem to indicate that there is no interaction with the tryptamine receptors.

Alkynes↗