NHE3 is the Na1yH1 exchanger located on the intestinal and renal brush border membrane, where it functions in transepithelial Na1 absorption. The brush border Na1 absorptive process is acutely inhibited by activation of cAMP-dependent protein kinase, but the molecular mechanism of this inhibitory effect is poorly understood. We have identified two regulatory proteins, E3KARP and NHERF, that interact with NHE3 to enable cAMP to inhibit NHE3. The two regulatory proteins are structurally related, sharing '50% identity in amino acid sequences. It has been previously shown that when NHE3 is transfected into PS120 fibroblasts or Caco-2 cells, cAMP failed to inhibit NHE3 activity. Northern blot analysis showed that both PS120 and Caco-2 cells lacked the expression of both E3KARP and NHERF. In contrast, other cell lines in which cAMP inhibits NHE3, including OK, CHO, and LLC-PK1 cells, expressed NHERF-related regulatory proteins. To determine their functions in cAMPdependent inhibition of NHE3, E3KARP and NHERF were transfected into PS120yNHE3 fibroblasts. Transfection in PS120yNHE3 fibroblasts with either NHERF or E3KARP reconstituted cAMP-induced inhibition of NHE3, resulting in 25–30% inhibition in these cells. Increased cAMP elicited by enterotoxins and some neurotransmitters results in stimulation of net Cl2 secretion and inhibition of electroneutralNaCl absorption in intestinal epithelium (1). Cl2 secretion and inhibition of NaCl absorption by the apical Na1yH1 exchanger primarily occur in the crypt and villus epithelial cells, respectively (1). Although inhibition of NaCl absorption and the apical Na1yH1 exchanger in the small intestine is a major contributor to diarrhea, very little is known about the molecular mechanisms of the cAMP-dependent inhibition of the absorptive process. Similarly, increased cAMP levels in renal proximal convoluted tubules are associated with inhibition of the apical membrane Na1yH1 exchanger (2). NHE3 is the small intestinal as well as renal proximal tubule brush border Na1yH1 exchanger that is involved in transepithelial NaCl and NaHCO3 absorption (see ref. 3 for review). All Na1yH1 exchangers respond to a variety of stimuli, including intracellular alkalinization, growth factors, hormones, tumor promoters, and hypertonic stress, and the different biological effects resulting from such stimuli are believed to be mediated by activation of protein kinases (1, 4). It was initially speculated that the regulation of Na1yH1 exchangers by these stimuli is mediated by phosphorylation of the exchanger. This speculation is based on the facts that most of the biological stimuli affecting Na1yH1 exchangers are believed to be mediated by protein kinases, and the regulation of Na1yH1 exchangers was shown to be dependent on the availability of cellular ATP (5–8). However, recent studies showed that the protein kinaseinduced regulation does not always correlate with changes in phosphorylation levels of the Na1yH1 exchanger and that phosphorylation of the Na1yH1 exchanger alone cannot account for its regulation (6, 9). Wakabayashi et al. (9) showed that deletion of all major phosphorylation sites in the cytoplasmic tail of NHE1 failed to completely obliterate the protein kinase stimulation. In addition, we have recently observed that the stimulation of NHE3 by fetal bovine serum and fibroblast growth factor and inhibition by phorbol ester in PS120 cells did not affect the phosphorylation level of NHE3 (J. Yip, M.D., C.T., unpublished data). These observation led to a hypothesis that, at least in some cases, Na1yH1 exchangers are regulated via accessory regulatory proteins that mediate the cellular signals between protein kinases and the exchangers. Therefore, we initiated a study to identify some of the proteins interacting with NHE3 by using a yeast two-hybrid system. One of the clones obtained, E3KARP, showed a high homology with the protein, NHERF, previously identified by Weinman et al. (11, 12). NHERF was identified by cellular fractionation of rabbit renal brush border vesicles, and has been shown to reconstitute cAMP-dependent inhibition of the brush border Na1yH1 exchanger in renal brush border vesicles in vitro (11). We show that both E3KARP and NHERF can reconstitute the cAMP-induced inhibition of NHE3 in a cell culture system, demonstrating the requirement for the presence of a regulatory protein. MATERIALS AND METHODS Yeast Two-Hybrid System. DNA encoding the entire cytoplasmic tail of NHE3 (amino acids 475–832) was cloned into the yeast LexA DNA-binding vector pEG202 (13). The resulting plasmid, pEG:C3, was used as a bait in two-hybrid screening of a WI-38 human lung fibroblast cDNA fused to the activation domain of GAL4 in the pJG4-5 plasmid. This cDNA library was kindly provided by Claude Sardet (Centre National de la Recherche Scientifique, Nice, France). To screen for interacting proteins, the yeast strain EGY48 was sequentially transfected with the bait plasmid and the cDNA library to obtain '3.5 3 106 primary transformants. Ninety-six of the primary transformants grew in the absence of leucine and had detectable b-galactosidase activity on 5-bromo-4-chloro-3indolyl b-D-galactoside (X-Gal) plates. Positive library plasThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y943010-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: VSVG, vesicular stomatitis virus glycoprotein; MBP, maltose-binding protein; GST, glutathione S-transferase; 8-Br-cAMP, 8-bromoadenosine 39,59-cAMP; PKA, cAMP-dependent protein kinase. †To whom reprint requests should be addressed at: The Johns Hopkins University School of Medicine, Department of Medicine, GI Unit, 918 Ross Building, 720 Rutland Avenue, Baltimore, MD 21205. e-mail: cyun@welchlink.welch.jhu.edu.