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      Characterization of Major Phosphoproteins in the cGMP-Mediated Protein Phosphorylation System of Vascular Smooth Muscle Membranes

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          Abstract

          G₀ (215–250 kD) and G<sub>1</sub> (120–140 kD), the unidentified major phosphoproteins in the cGMP-mediated protein phosphorylation system of vascular smooth muscle membranes, were compared for biochemical and immunological properties with the type 1 inositol 1,4,5-trisphosphate receptor (InsP<sub>3</sub>R, 240 kD) and the myosin-binding subunit (MBS, 138 kD) of myosin phosphatase, both of them substrates for cGMP-dependent protein kinase. Two microsomal proteins that were immunoreactive with antibodies to InsP<sub>3</sub>R and MBS were detected, and comigrated with G₀ and G<sub>1</sub>, respectively, on SDS-PAGE. When thiophosphorylated G₀ and G<sub>1</sub> were subjected to immunoprecipitation, MBS antibody induced the precipitation of a 138-kD phosphoprotein, but did not significantly affect the amount of G<sub>1</sub> remaining in the supernatant, while InsP<sub>3</sub>R antibody precipitated G₀ almost completely. Unexpectedly, InsP<sub>3</sub>R antibody coprecipitated a large portion of G<sub>1</sub>, which did not cross-react with either antibody to MBS or InsP<sub>3</sub>R. Just like InsP<sub>3</sub>R, G₀ bound to the calmodulin column in a Ca<sup>2+</sup>-dependent manner, and, again, a large portion of G<sub>1</sub> was copurified with G₀. These results suggest that G₀ is identical to InsP<sub>3</sub>R, while G<sub>1</sub> consists of several phosphoproteins, including the 138-kD protein associated with InsP<sub>3</sub>R as a major component. MBS is not G<sub>1</sub> or may represent only a minor component of it.

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          Most cited references 3

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          Cyclic GMP causes Ca2+ desensitization in vascular smooth muscle by activating the myosin light chain phosphatase.

           M R Lee,  L. Li,  T Kitazawa (1997)
          Using permeabilized, arterial smooth muscle strips where membrane-associated pathways remain intact but intracellular Ca2+ stores are depleted, we investigated mechanism(s) for the Ca2+ desensitization of contractile force by cGMP. The nonhydrolyzable analog 8-bromo-cGMP, when applied to these strips with submaximal Ca2+ levels clamped, dramatically and reversibly reduced the steady state levels of phosphorylation at 20-kDa myosin light chain and contractile force, with a nanomolar concentration required to obtain 50% reduction. Supramaximal concentrations of 8-bromo-cGMP (10 microM), however, did not change the steady state relationship between phosphorylation and force. When light chain phosphatase activity was blocked at pCa 6.7, 10 microM 8-bromo-cGMP did not affect the rates of rise of light chain phosphorylation and contractile force. When light chain kinase activity was blocked, 10 microM 8-bromo-cGMP significantly accelerated light chain dephosphorylation and force relaxation from the maximal contraction steady state. The light chain phosphorylation time course of a pCa 6. 0-induced contraction in the presence of 8-bromo-cGMP exhibited kinetics that are predictable from a mathematical model in which only light chain phosphatase activity is increased. The results of this study strongly suggest that cGMP indirectly activates light chain phosphatase, the first proposed mechanism for cGMP-induced Ca2+ desensitization in vasodilatation.
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            Molecular cloning of cDNA encoding the 110 kDa and 21 kDa regulatory subunits of smooth muscle protein phosphatase 1M.

            The structures of the M110 and M21 regulatory subunits of protein phosphatase-1M, the major enzyme which dephosphorylates myosin in smooth muscle, have been deduced from cloned cDNAs. The N-terminus of the M110 subunit from rat aorta contains seven ankyrin repeats, while the C-terminus of the M21 subunit from chicken gizzard contains a leucine zipper motif. The M110 subunit is expressed in two different forms which differ in their C-terminal sequences. One of these is highly homologous to the whole of the M21 subunit.
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              • Record: found
              • Abstract: not found
              • Article: not found

              Phosphorylation of the Inositol 1,4,5-Trisphosphate Receptor: CYCLIC GMP-DEPENDENT PROTEIN KINASE MEDIATES cAMP AND cGMP DEPENDENT PHOSPHORYLATION IN THE INTACT RAT AORTA

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                Author and article information

                Journal
                JVR
                J Vasc Res
                10.1159/issn.1018-1172
                Journal of Vascular Research
                S. Karger AG
                1018-1172
                1423-0135
                1999
                August 1999
                27 August 1999
                : 36
                : 4
                : 299-310
                Affiliations
                aDepartment of Pharmacology and bFirst Department of Internal Medicine, Niigata University School of Medicine, Niigata, and cFirst Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie, Japan
                Article
                25658 J Vasc Res 1999;36:299–310
                10.1159/000025658
                10474043
                © 1999 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 8, References: 42, Pages: 12
                Categories
                Research Paper

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