Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.cholesterol ͉ intestinal brush border membranes

Many receptors that couple to heterotrimeric G proteins have been shown to mediate the rapid activation of MAP 1 kinases. Among these are receptors for several substances either present in the general circulation, released as neurotransmitters, or produced locally by vascular endothelium or activated platelets. These include catecholamines, acetylcholine, pituitary glycopeptide hormones, adenosine, angiotensins, bombesin, endothelins, LPA, and ␣-thrombin (1). Receptors for these substances, activated in response to systemic or locally generated ligands, may in turn play significant roles in the endocrine or paracrine regulation of cell proliferation.Heterogeneity exists in the mechanisms whereby G proteincoupled receptors activate MAP kinases. Depending upon receptor and cell type, MAP kinase activation may be mediated by pertussis toxin-sensitive or -insensitive G proteins and be either PKC-or Ras-dependent. In COS-7 cells, for example, activation of MAP kinase via the pertussis toxin-insensitive, Gq-coupled, ␣1B adrenergic and M1 muscarinic acetylcholine receptors is significantly inhibited by PKC depletion but insensitive to expression of a dominant-negative mutant of Ras. In contrast, activation of MAP kinase via the pertussis toxinsensitive Gi-coupled ␣2A adrenergic and M2 muscarinic acetylcholine receptors is PKC-independent but requires Ras activation and is sensitive to inhibitors of tyrosine protein kinases (2). Similarly, LPA, a potent stimulator of mitogenesis in quiescent fibroblasts that signals via a G protein-coupled receptor coupling to both pertussis toxin-sensitive and -insensitive G proteins (3-5), activates MAP kinase via a pertussis toxin-sensitive pathway involving Ras and Raf activation (6, 7). LPA-mediated MAP kinase activation is sensitive to tyrosine kinase inhibitors (7, 8) but independent of its effects on phosphatidylinositol hydrolysis and its ability to inhibit adenylyl cyclase (4,8). In COS-7 cells, Ras-dependent MAP kinase activation via ␣2A adrenergic (9), M2 muscarinic acetylcholine, D2 dopamine, and A1 adenosine receptors (10) is mediated largely by G␤␥ subunits derived from pertussis toxin-sensitive G proteins. Indeed, overexpression of G␤␥ subunits, but not constitutively activated G␣i1 or G␣i2 mutants, is sufficient to activate MAP kinase (9 -11) in these cells.

Receptors that couple to the heterotrimeric G proteins, Gi or Gq, can stimulate phosphoinositide (PI) hydrolysis and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC), which can stimulate increased MAPK activity. However, the relationship between PI hydrolysis and MAPK activation in Gi and Gq signaling has not been clearly defined and is the subject of this study. The effects of several signaling inhibitors are assessed including expression of a peptide derived from the carboxyl terminus of the beta adrenergic receptor kinase 1 (beta ARKct), which specifically blocks signaling mediated by the beta gamma subunits of G proteins (G beta gamma), expression of dominant negative mutants of p21ras (RasN17) and p74raf-1 (N delta Raf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC. The Gi-coupled alpha 2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of beta ARKct, RasN17, or N delta Raf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the Gq-coupled alpha 1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of beta ARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of N delta Raf or by PKC depletion. These data demonstrate that Gi- and Gq-coupled receptors stimulate MAPK activation via distinct signaling pathways. G beta gamma is responsible for mediating Gi-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, G alpha mediates Gq-coupled receptor-stimulated MAPK activation using a p21ras-independent mechanism employing PKC and p74raf. To define the role of G beta gamma in Gi-coupled receptor-mediated PI hydrolysis and MAPK activation, direct stimulation with G beta gamma was used. Expression of G beta gamma resulted in MAPK activation that was sensitive to inhibition by expression of beta ARKct, RasN17, or N delta Raf or by PTK inhibitors, but insensitive to PKC depletion. By comparison, G beta gamma-mediated PI hydrolysis was not affected by beta ARKct, RasN17, or N delta Raf expression or by PTK inhibitors. Together, these results demonstrate that G beta gamma mediates MAPK activation and PI hydrolysis via independent signaling pathways.