CHARACTERIZATION OF THE GAP JUNCTION PROTEIN, CONNEXIN45

Connexin45 is a gap junction protein which forms channels with unique characteristics. RNA blots demonstrated that connexin45 is expressed i n a number of cell lines including WB, SK Hep1, BHK, A7r5, CLEM, and B WEM cells. Connexin45 was further studied in BWEM cells using specific affinity-purified antibodies directed against a synthetic peptide rep resenting amino acids 285-298 of its sequence. Immunofluorescence expe riments demonstrated that the BWEM cells expressed both connexin43 and connexin45 and that these connexins colocalized. Connexin45 polypepti de, immunoprecipitated from BWEM cells metabolically labeled with [S-3 5]-methionine, consisted of a predominant 48 kD polypeptide. Connexin4 5 and connexin43 contained radioactive phosphate when immunoprecipitat ed from BWEM cells metabolically labeled with [P-32]-orthophosphoric a cid. This phosphate label was removed from connexin45 by alkaline phos phatase digestion. Treatment of BWEM cells with the tumor promoting ag ent 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited intercellular passage of microinjected Lucifer yellow. While TPA treatment induced phosphorylation of connexin43 in these cells, it reduced the expressio n of connexin45. Furthermore, the connexin45 expressed after TPA treat ment was not phosphorylated. These results suggest that treatments whi ch alter protein phosphorylation may regulate connexin43 and connexin4 5 in BWEM cells by different mechanisms.