Topography of connexin32 in rat liver gap junctions. Evidence for an intramolecular disulphide linkage connecting the two extracellular peptide loops.
A range of anti-peptide antibodies directed towards selected amino acid sequences of connexin32 was prepared and characterised. The site-directed antibodies that identified connexin32 were used to study by immunolocalization and by proteolytic treatment of intact and split gap junctions the arrangement of the protein in the membrane. These studies reinforce models of connexin topography in which the polypeptide traverses the junctional membrane four times, with the amino and carboxyl termini cytoplasmically located. The four transmembrane domains were shown to be linked by two extracellular loops with a single intracellular loop connecting the second and third transmembrane domains. Evidence is presented to show that the two extracellular domains of connexin32, which are important for intercellular adhesion and the insulated bridging of the extracellular space by channels allowing cell-cell communication across the gap junction, are connected by disulphide bond(s). The studies lead to a more detailed two-dimensional model of connexin32 in the membrane, incorporating the favoured theoretical arrangement of disulphide bonds at the extracellular domain of connexin32.
Rahman
S
S
Laboratory of Protein Structure, National Institute of Medical Research, Mill Hill, London, UK.
Evans
W H
WH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D017630:Connexins; D004220:Disulfides / Q000378:metabolism; D007150:Immunohistochemistry; D007365:Intercellular Junctions / Q000378:metabolism* / Q000648:ultrastructure; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011487:Protein Conformation; D051381:Rats
D000906:Antibodies; D017630:Connexins; D004220:Disulfides; D008565:Membrane Proteins; D010446:Peptide Fragments