SELECTIVE MONOCLONAL-ANTIBODY RECOGNITION AND CELLULAR-LOCALIZATION OF AN UNPHOSPHORYLATED FORM OF CONNEXIN43

A sequence-specific monoclonal antibody directed against the gap junct ion protein connexin43 (Cx43) is shown here to be specific for the unp hosphorylated form of this protein. In tissues and cultured cells cont aining different phosphorylated and unphosphorylated forms of Cx43, th e antibody detected only the latter as shown by Western blotting of na tive and alkaline phosphatase-treated samples. Immunohistochemically, this monoclonal antibody did not recognize gap junctions in the vast m ajority of cultured cardiac myocytes, where nearly all detectable Cx43 is phosphorylated. In contrast, it was able to detect some intracellu lar Cx43 in tracheal smooth muscle cells and an epithelial cell line ( Cl-9 cells), producing patterns of labeling consistent with those seen using a polyclonal antibody that recognizes both phosphorylated and u nphosphorylated forms of Cx43. Immunostaining of gap junctions in the cultured cells indicates that both phosphorylated and unphosphorylated Cx43 are present in some assembled gap junctions, suggesting that ass embled junctions do not contain exclusively the phosphorylated form of the protein. Annular gap junctions, believed to form as part of the p athway for internalization and degradation of gap junctions, were only occasionally and sparsely labeled by the monoclonal antibody, indicat ing that complete protein dephosphorylation is not required for uptake and degradation of gap junctions. Furthermore, the ability of this an tibody to recognize only unphosphorylated Cx43, and not any of the pho sphorylated forms present in the tissues and cell types examined, sugg ests that a unique phosphorylation site, perhaps present in the epitop e recognized by this antibody, must be phosphorylated prior to phospho rylation of Cx43 at other sites. (C) 1997 Academic Press.