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.
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242, USA.
F J
FJ
McDonald
M J
MJ
Welsh
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