Water channel vesicles from toad urinary bladder contain a family of proteins present in other tissues.
Antidiuretic hormone (ADH) stimulation causes the fusion and subsequent retrieval of cytoplasmic vesicles containing water channels (WCV) with the apical membrane of toad bladder granular cells. Previously, we showed that purified WCV contain 12 major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To identify various WCV proteins, we screened a panel of mouse monoclonal antibodies and characterized an immunoglobulin G1 monoclonal antibody, 5E5, that recognizes integral membrane WCV proteins of 38, 33, and 31 kDa. Immunocytochemistry and Western blot analyses show that 5E5 binds to multivesicular body endosomes shown previously to contain ADH water channels. In addition, 5E5 recognizes these proteins in selected cells of the skin, intestine, liver, kidney, spleen, and lung. However, 5E5 does not appear to recognize components of the water channel itself. We conclude that WCV contain several membrane proteins recognized by 5E5 that are present in certain cells of the other organs. Monoclonal 5E5 provides a probe to determine the structure and function of these endosomal proteins as well as their role in the ADH water permeability response.
Paredes
A
A
Division of Nephrology, Children's Hospital, Boston, Massachusetts 02030.
Briscoe
D M
DM
Williams
C K
CK
Garcia-Perez
A
A
Wade
J B
JB
Harris
H W
HW
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002025:Bufonidae; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007473:Ion Channels / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D009928:Organ Specificity; D011817:Rabbits; D051381:Rats; D013045:Species Specificity; D001743:Urinary Bladder / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D007473:Ion Channels; D008565:Membrane Proteins