CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to decrease [corrected] plasma membrane localization and 86Rb+ uptake.
The carboxy terminus (CT) of the colonic H(+)-K(+)-ATPase is required for stable assembly with the beta-subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HKalpha(2), we selected 84 amino acids in the CT of the alpha-subunit of mouse colonic H(+)-K(+)-ATPase (CT-HKalpha(2)) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HKalpha(2) with CD63, recombinant CT-HKalpha(2) and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HKalpha(2) protein (but not CT-HKalpha(1)) coprecipitated with CD63, confirming stable assembly of HKalpha(2) with CD63. In HEK-293 transfected with HKalpha(2) plus beta(1)-Na(+)-K(+)-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKalpha(2)/NKbeta(1) and (86)Rb(+) uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HKalpha(2)-NKbeta(1) complex in the cell membrane.
Codina
Juan
J
Section of Nephrology, Department of Internal Medicine, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA.
Li
Jian
J
Dubose
Thomas D
TD
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D003106:Colon / Q000201:enzymology*; D015870:Gene Expression / Q000502:physiology; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000201:enzymology; D051379:Mice; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D012414:Rubidium Radioisotopes; D012441:Saccharomyces cerevisiae; D060149:Tetraspanin 30
D015703:Antigens, CD; C554586:CD63 protein, human; C554587:Cd63 protein, mouse; D010980:Platelet Membrane Glycoproteins; D012414:Rubidium Radioisotopes; D060149:Tetraspanin 30; D017506:H(+)-K(+)-Exchanging ATPase