NHE3 serves as a molecular tool for cAMP-mediated regulation of receptor-mediated endocytosis.
Receptor-mediated, clathrin-dependent endocytosis (RME) is important for macromolecular transport and regulation of cell-surface protein expression. Pharmacological studies have shown that the plasma membrane transport protein Na(+)/H(+) exchanger 3 (NHE3), which shuttles between the plasma membrane and the early endosomal compartment by means of clathrin-mediated endocytosis, contributes to endosomal pH homeostasis and endocytic fusion events. Furthermore, it is known that NHE3 is phosphorylated and inhibited by cAMP-dependent kinase (protein kinase A). Here, we show, in a cellular knockout/retransfection approach, that NHE3 supports RME and confers cAMP sensitivity to RME, using megalin/cubilin-mediated albumin uptake in opossum kidney cells. RME, but not fluid-phase endocytosis, was dependent on NHE3 activity and expression. Furthermore, NHE3 deficiency or inhibition reduced the relative surface expression of megalin without altering total expression. In wild-type cells, cAMP inhibits NHE3 activity, leads to endosomal alkalinization, and reduces RME. In NHE3-deficient cells, endosomal pH is not sensitive to NHE3 inhibition, and cAMP does not affect endosomal pH or RME. NHE3 transfection into deficient cells restores RME and the effects of cAMP. Thus our data show that NHE3 is important for cAMP sensitivity of clathrin-dependent RME.
Gekle
Michael
M
Physiologisches Institut, University of Würzburg, 97070 Würzburg, Germany. michael.gekle@mail.uni-wuerzburg.de
Serrano
Oscar K
OK
Drumm
Karina
K
Mildenberger
Sigrid
S
Freudinger
Ruth
R
Gassner
Birgit
B
Jansen
Hans Willi
HW
Christensen
Erik I
EI
D000418:Albumins / Q000378:metabolism; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D002966:Clathrin / Q000502:physiology*; D000242:Cyclic AMP / Q000502:physiology*; D004705:Endocytosis / Q000502:physiology*; D006706:Homeostasis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism*; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000032:analysis / Q000502:physiology; D016296:Mutagenesis; D009893:Opossums; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics* / Q000502:physiology*; D014162:Transfection
D000418:Albumins; D002966:Clathrin; D026561:Low Density Lipoprotein Receptor-Related Protein-2; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride; D003994:Bucladesine; D000242:Cyclic AMP