We recently reported that megalin is subjected to regulated intramembrane proteolysis (RIP) and includes: (1) protein kinase C (PKC)-regulated, metalloprotease-mediated ectodomain shedding producing a membrane bound megalin C-terminal fragment (MCTF) and, (2) -secretase-mediated cleavage of the MCTF producing a soluble megalin intracellular domain (MICD). Based on studies of RIP of other receptors, the MICD is predicted to target to the nucleus and regulate gene expression. In order to determine if RIP of megalin regulates proximal tubule gene expression we stably expressed the transfected MCTF (tMCTF) or transfected MICD (tMICD) in opossum kidney proximal tubule (OKP) cells and examined the resulting phenotype. Immunoblotting and immunocytochemical analysis of tMCTF cells showed the tMCTF was expressed and constitutively processed by -secretase. Analysis of specific protein expression in tMCTF- and tMICD-transfected cells using western blot showed endogenous megalin and Na⁺/H⁺ exchanger 3 (NHE3) protein expression to be dramatically lower than that of control cells. Expression of other proteins including myosin VI, -adaptin, and the Na, K-ATPase, appeared unchanged. Analysis of specific mRNA expression using quantitative real-time PCR showed megalin and NHE3 mRNA levels were significantly lower in tMCTF- and tMICD-transfected cells compared to controls. Inhibition of -secretase activity in tMCTF cells resulted in an 8-10-fold recovery of megalin mRNA within four hours. These data show that the C-terminal domain of megalin regulates expression of specific proteins in OKP cells and provides the first evidence that RIP of megalin may be part of a signaling pathway linking protein absorption and gene expression in proximal tubule.