The low density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional cell surface receptor that interacts through its cytoplasmic tail with adaptor and scaffold proteins that participate in cellular signaling. Its extracellular domain, like that of the signaling receptor Notch and of amyloid precursor protein (APP), is proteolytically processed at multiple positions. This similarity led us to investigate whether LRP, like APP and Notch, might also be cleaved at a third, intramembranous or cytoplasmic site, resulting in the release of its intracellular domain. Using independent experimental approaches we demonstrate that the cytoplasmic domain is released by a ␥-secretase-like activity and that this event is modulated by protein kinase C. Furthermore, cytoplasmic adaptor proteins that bind to the LRP tail affect the subcellular localization of the free intracellular domain and may regulate putative signaling functions. Finally, we show that the degradation of the free tail fragment is mediated by the proteasome. These findings suggest a novel role for the intracellular domain of LRP that may involve the subcellular translocation of preassembled signaling complexes from the plasma membrane.Seven structurally closely related cell surface receptors constitute the core of the low density lipoprotein (LDL) 1 receptor gene family. They include the LDL receptor, the LDL receptorrelated protein (LRP or LRP1), LRP1b, megalin, very low density lipoprotein (VLDL) receptor, apolipoprotein E receptor 2 (apo-ER2), and MEGF7 (multiple epidermal growth factor-like domains containing protein 7) (1). Although the members of this evolutionarily ancient gene family all share the same typical arrangement of protein modules in their extracellular domains, the biological functions of the individual members of the family are highly diverse and include roles in cellular ligand uptake and endocytosis, the transmission of extracellular signals, vitamin and cholesterol homeostasis, brain development, the modulation of neurotransmission, and protection from neurodegeneration (1, 2).Of this entire receptor family, LRP interacts with by far the largest number of proteins (3). Ligands that bind to its extracellular domain include, for instance, ␣2-macroglobulin, plasminogen activators, clotting factors, lipases, and the amyloid precursor protein (APP). The cytoplasmic tail of LRP also interacts with an extended set of intracellular adaptor and scaffold proteins, e.g. Dab1, c-Jun amino-terminal kinase interacting proteins, the postsynaptic density protein PSD-95 (4), and the trivalent scaffold protein FE65 (5). The latter protein contains two phosphotyrosine binding domains, of which the first binds to the LRP tail, whereas the second domain interacts with an NPXY motif in the cytoplasmic tail of APP (6 -9).Mice in which the LRP gene has been disrupted by homologous recombination die early during embryonic development (10). The phenotype of the few malformed LRP-deficient embryos that survive until around E9 -E10 is complex and ...

Abstract-Endothelium-dependent hyperpolarization and relaxation of vascular smooth muscle are mediated by endothelium-derived hyperpolarizing factors (EDHFs). EDHF candidates include cytochrome P-450 metabolites of arachidonic acid, K ϩ , hydrogen peroxide, or electrical coupling through gap junctions. In bovine coronary arteries, epoxyeicosatrienoic acids (EETs) appear to function as EDHFs. A 14,15-EET analogue, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) was synthesized and identified as an EET-specific antagonist. In bovine coronary arterial rings preconstricted with U46619, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET induced concentration-related relaxations. Preincubation of the arterial rings with 14,15-EEZE (10 mol/L) inhibited the relaxations to 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET but was most effective in inhibiting 14,15-EET-induced relaxations. 14,15-EEZE also inhibited indomethacin-resistant relaxations to methacholine and arachidonic acid and indomethacin-resistant and L-nitroarginineresistant relaxations to bradykinin. It did not alter relaxation responses to sodium nitroprusside, iloprost, or the K ϩ channel activators (NS1619 and bimakalim). Additionally, in small bovine coronary arteries pretreated with indomethacin and L-nitroarginine and preconstricted with U46619, 14,15-EEZE (3 mol/L) inhibited bradykinin (10 nmol/L)-induced smooth muscle hyperpolarizations and relaxations. In rat renal microsomes, 14,15-EEZE (10 mol/L) did not decrease EET synthesis and did not alter 20-hydroxyeicosatetraenoic acid synthesis. This analogue acts as an EET antagonist by inhibiting the following: (1) EET-induced relaxations, (2) the EDHF component of methacholineinduced, bradykinin-induced, and arachidonic acid-induced relaxations, and (3) the smooth muscle hyperpolarization response to bradykinin. Thus, a distinct molecular structure is required for EET activity, and alteration of this structure modifies agonist and antagonist activity. These findings support a role of EETs as EDHFs. Key Words: epoxyeicosatrienoic acids Ⅲ arachidonic acid Ⅲ endothelium-derived hyperpolarizing factors Ⅲ endothelium Ⅲ 20-hydroxyeicosatetraenoic acid E poxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachidonic acid. They are synthesized by the vascular endothelium and released in response to vasoactive agonists, such as bradykinin and acetylcholine, and are stimulated by cyclic stretch. [1][2][3][4][5][6] Additionally, in the coronary circulation, they cause vascular smooth muscle hyperpolarization and relaxation and, therefore, function as endothelium-derived hyperpolarizing factors (EDHFs). 2 To investigate the role of endogenous EETs in the relaxations induced by endothelium-dependent vasoactive substances, inhibitors of cytochrome P-450 are used, but these investigations have given variable results. In some studies, inhibitors of cytochrome P-450 block the relaxations to bradykinin and acetylcholine, whereas in other studies, these inhibitors are without effect or also inhibit relaxations to K ϩ ...

Epoxyeicosatrienoic acids (EETs) are endothelium-derived eicosanoids that activate potassium channels, hyperpolarize the membrane, and cause relaxation. We tested 19 analogs of 14,15-EET on vascular tone to determine the structural features required for activity. 14,15-EET relaxed bovine coronary arterial rings in a concentration-related manner (ED(50) = 10(-6) M). Changing the carboxyl to an alcohol eliminated dilator activity, whereas 14,15-EET-methyl ester and 14,15-EET-methylsulfonimide retained full activity. Shortening the distance between the carboxyl and epoxy groups reduced the agonist potency and activity. Removal of all three double bonds decreased potency. An analog with a Delta8 double bond had full activity and potency. However, the analogs with only a Delta5 or Delta11 double bond had reduced potency. Conversion of the epoxy oxygen to a sulfur or nitrogen resulted in loss of activity. 14(S),15(R)-EET was more potent than 14(R),15(S)-EET, and 14,15-(cis)-EET was more potent than 14,15-(trans)-EET. These studies indicate that the structural features of 14,15-EET required for relaxation of the bovine coronary artery include a carbon-1 acidic group, a Delta8 double bond, and a 14(S),15(R)-(cis)-epoxy group.

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