Molecular cloning of a regulatory protein for membrane-bound guanylate cyclase GC-A

Biochem Biophys Res Commun. 2000 Nov 11;278(1):106-11. doi: 10.1006/bbrc.2000.3761.

Abstract

Activation of membrane-bound guanylate cyclase GC-A by atrial natriuretic factor (ANF) may require the involvement of accessory proteins. To identify these postulated proteins, we isolated a 1. 0-kb cDNA clone from a rat brain expression library using a polyclonal antiserum against mastoparan. The 1.0-kb cDNA encodes a protein of 111 amino acids. Expression of this cDNA in COS-7 cells potentiated ANF-stimulated GC-A activity. Therefore, the 1.0-kb gene encodes a guanylate cyclase regulatory protein (GCRP). Fluorescence microscopy studies using the fusion protein of GCRP with green fluorescence protein (GFP) indicated that GCRP was present in the cytosol in PC12 cells, but translocated toward the plasma membrane in the presence of ANF. Coimmunoprecipitation experiments indicate that GCRP associates with GC-A in the presence of ANF. These results suggest that ANF induces the association of GCRP with GC-A and this association contributes to the activation of GC-A.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / chemistry
  • Animals
  • Base Sequence
  • Blotting, Western
  • Brain / metabolism
  • CHO Cells
  • COS Cells
  • Cell Membrane / metabolism
  • Cloning, Molecular
  • Cricetinae
  • Cyclic GMP / metabolism
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Enzyme Activation
  • Gene Library
  • Green Fluorescent Proteins
  • Guanylate Cyclase / genetics*
  • Guanylate Cyclase / metabolism*
  • Intercellular Signaling Peptides and Proteins
  • Intracellular Signaling Peptides and Proteins
  • Luminescent Proteins / metabolism
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • PC12 Cells
  • Peptides
  • Plasmids / metabolism
  • Precipitin Tests
  • Protein Binding
  • Proteins / genetics*
  • Rats
  • Receptors, Enterotoxin
  • Receptors, Guanylate Cyclase-Coupled
  • Receptors, Peptide / genetics*
  • Recombinant Fusion Proteins / metabolism
  • Tissue Distribution
  • Translocation, Genetic
  • Wasp Venoms / immunology

Substances

  • Amino Acids
  • DNA, Complementary
  • Intercellular Signaling Peptides and Proteins
  • Intracellular Signaling Peptides and Proteins
  • Luminescent Proteins
  • Peptides
  • Proteins
  • Receptors, Peptide
  • Recombinant Fusion Proteins
  • Rundc3a protein, rat
  • Wasp Venoms
  • Green Fluorescent Proteins
  • mastoparan
  • Guanylate Cyclase
  • Receptors, Enterotoxin
  • Receptors, Guanylate Cyclase-Coupled
  • Cyclic GMP