Binding of the proline-rich region of the epithelial Na+ channel to SH3 domains and its association with specific cellular proteins.
The amiloride-sensitive epithelial Na+ channel (ENaC) is an important component of the Na(+)-reabsorption pathway in many epithelia. The identification of three subunits of ENaC (alpha, beta and gamma), as well as results from a number of functional and biochemical studies, suggests that functional Na+ channels are composed of a complex of proteins. To learn about possible interactions of the channel with other proteins, we studied the alpha-subunit of rat and human ENaC. We found that the proline-rich C-terminal domains of both rat and human alpha-ENaC, expressed as glutathione S-transferase fusion proteins, bound to SH3 domains in vitro. A 116 kDa protein from a human lung adenocarcinoma cell line (H441) was specifically bound by the human alpha-ENaC C-terminal fusion protein and by a shorter 18-amino acid proline-rich peptide derived from the larger fusion protein. The 116 kDa protein was not glycosylated and was not phosphorylated on tyrosine or by cyclic AMP-dependent protein kinase (PKA). A 134 kDa protein which was also bound by the human alpha-ENaC C-terminal fusion protein was a substrate for phosphorylation by PKA. These data suggest that the proline-rich C-terminal tail of alpha-ENaC may interact with other proteins that control its function, regulation or localization.
McDonald
F J
FJ
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242, USA.
Welsh
M J
MJ
D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D002460:Cell Line; D004848:Epithelium / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis; D006801:Humans; D008175:Lung Neoplasms; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011392:Proline; D015531:Radioimmunoprecipitation Assay; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015222:Sodium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured; D018909:src Homology Domains
D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; D013462:Sulfur Radioisotopes; D011392:Proline; D008715:Methionine; D005982:Glutathione Transferase