It has recently been shown that the epithelial Na؉ channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the ␤-and ␥-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel.Amiloride-sensitive epithelial Na ϩ channels (ENaC) 3 are membrane proteins that are expressed in salt-absorptive epithelia, including the distal collecting tubules of the kidney, the mucosa of the distal colon, the respiratory epithelium, and the excretory ducts of sweat and salivary glands (1-4). Na ϩ absorption via ENaC is critical to the normal regulation of Na ϩ and fluid homeostasis and is important for maintaining blood pressure (5) and the volume of fluid in the respiratory passages (6). Increased ENaC activity has been implicated in the salt-sensitive inherited form of hypertension, Liddle's syndrome (7), and dehydration of the surface of the airway epithelium in the pathology associated with cystic fibrosis lung disease (8).Expression of ENaC at the cell membrane surface is regulated by the E3 ubiquitin protein ligase, Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4) (9).Interaction between the WW domains of Nedd4-2 and the proline-rich PY motifs (PPPXY) on ENaC is essential for Nedd4-2 to exert a negative effect on the channel (10, 11). This interaction leads to ubiquitination-dependent internalization of ENaC (12, 13). Several regulators of ENaC exert their effects on the channel by modulating the action of Nedd4-2. For instance, serum and glucocorticoid-dependent protein kinase (14), protein kinase B (15), and G protein-coupled receptor kinase (16) up-regulate activity of ENaC by inhibiting Nedd4-2. Although the details of cellular mechanisms that underlie internalization of ENaC remain to be elucidated, the physiological significance of Nedd4-dependent internalization of the channel has been well established. For instance, heritable mutations that delete the cytosolic termini of the ␤-or ␥-subunit of ENaC, which contain the proline-rich motifs, are known to cause hyperactivity of ENaC in the kidney (17) and increase cell surface expression of the channel (7, 18).The plasma membranes of most cell t...

Neurotensin (NT) receptors are overexpressed in different human tumors, such as human ductal pancreatic adenocarcinoma. New stable neurotensin analogs with high receptor affinity have been synthesized by replacing arginine residues with lysine and arginine derivatives. The aim of this study was to explore the biodistribution, tumor uptake, kidney localization, and stability characteristics of these new analogs in order to develop new diagnostic tools for exocrine pancreatic cancer. Four (111)In-labeled DTPA-chelated NT analogs and one (111)In-labeled DOTA-chelated NT analog were evaluated in NMRI nude mice bearing NT receptor-positive HT29 tumors. Experiments with a coinjection of unlabeled NT or lysine were performed to investigate receptor-mediated uptake and kidney protection, respectively. In addition, the in vivo serum stability of the most promising analog was analyzed. In the biodistribution study in mice, at 4 hours postinjection, a low percentage of the injected dose per gram (%ID/g) of tissue for all compounds was found in NT receptor-negative organs, such as the blood, spleen, pancreas, liver, muscle, and femur. A high uptake was found in the colon, intestine, kidneys, and in implanted HT29 tumors. The coinjection of excess unlabeled neurotensin significantly reduced tumor uptake, showing tumor uptake to be receptor-mediated. To a lesser extent, this was also observed for the colon, but not for other tissues. We concluded that DTPA-(Pip)Gly-Pro-(PipAm)Gly-Arg-Pro-Tyr-tBuGly-Leu-OH and the DOTA-linked counterpart have the most favorable biodistribution properties regarding tumor uptake.

The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with ␣-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na ؉ absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant ( K220R GRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (⌬RH-GRK2) or a GRK2 mutant that cannot interact with G␣q/11/14 ( D110A GRK2), increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the ␣-subunits of Gq/11. We further found that expression of constitutively active G␣q/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against G␣q/11 increases ENaC activity. The effect of G␣q on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 ␣-subunits regulate the activity ENaC.The ␤-adrenergic receptor kinase 1 (GRK2) is one of the G protein-coupled receptor serine/threonine kinases (GRKs). 3All seven members of the GRK family (GRK1-7) share a highly homologous kinase domain that is flanked toward the C-terminal by a pleckstrin homology (PH) domain and toward the N-terminal by a regulator of G-protein signaling homology (RH) domain (1). Protein kinases of this family are unique in their ability to specifically phosphorylate the agonist-activated form of heptahelical G protein-coupled receptors (GPCRs) (2). Upon stimulation, GRKs facilitate binding of agonist-activated GPCRs to cytosolic cofactor proteins, arrestins, and other proteins involved in receptor desensitization (3,4). This interaction impairs coupling between the GPCRs and trimeric G proteins, targets GPCRs for clathrin-mediated endocytosis (3), and terminates G protein signal transduction (2). Activity of GRKs is, therefore, important for arbitrating an appropriate strength and duration of cellular responses, allowing the G protein-dependent cellular responses to physiological stimulation to cease rapidly after receptor activation even in the continuing presence of stimuli.Recent studies suggest that, in addition to their ability to inactivate GPCRs by phosphorylation, GRKs can phosphorylate and regulate activity of an array of non-receptor substrates (2) and also regulate GPCR signaling by a mechanism that does not require their intrinsic kinase activity (3). For instance, the RH domain of GRK2 contains a binding site th...