C. Schroeder

NOSIP, a novel modulator of endothelial nitric oxide synthase activity

Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector proteins such as Ca2+‐calmodulin, by posttranslational modifications such as phosphorylation via protein kinase B, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of eNOS is thought to contribute to the intracellular trafficking of the enzyme;however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two‐hybrid system and identified a novel 34 kDa protein, termed NOSIP (eNOS interacting protein), which avidly binds to the carboxyl‐terminal region of the eNOS oxygenase domain. Coimmunoprecipitation studies demonstrated the specific interaction of eNOS and NOSIP in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the caveolin‐1 scaffolding domain. NO production was significantly reduced in eNOS‐expressing CHO cells (CHO‐eNOS) that transiently overexpressed NOSIP. Stimulation with the calcium ionophore A23187 induced the reversible translocation of eNOS from the detergentinsoluble to the detergent‐soluble fractions of CHO‐eNOS, and this translocation was completelyprevented by transient coexpression of NOSIP in CHO‐eNOS. Immunofluorescence studies revealed a prominent plasma membrane staining for eNOS in CHO‐eNOS that was abolished in the presence of NOSIP. Subcellular fractionation studies identified eNOS in the caveolin‐rich membrane fractions of CHO‐eNOS, and coexpression of NOSIP caused a shift of eNOS to intracellular compartments. We conclude that NOSIP is a novel type of modulator that promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis.—Dedio, J., König, P., Wohlfart, P., Schroeder, C., Kummer, W., Muller‐Esterl, W. NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J. 15, 79–89 (2001)

Palmitoylation of Endothelin Receptor A DIFFERENTIAL MODULATION OF SIGNAL TRANSDUCTION ACTIVITY BY POST-TRANSLATIONAL MODIFICATION

Post-translational modifications such as phosphorylation and palmitoylation play important roles for the function and regulation of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins. Here we demonstrate that the human endothelin receptor A (ETA) incorporates [3H]palmitate. Mutation of a cluster of five cysteine residues present in the cytoplasmic tail of ETA into serine or alanine residues completely prevented palmitoylation of the receptor. The ligand binding affinity of the non-palmitoylated ETA mutants was essentially unchanged as compared to the palmitoylated wild type ETA suggesting that the replacement of the cysteine residues did not alter the overall structure of the receptor. Furthermore, the ligand-induced stimulation of adenylyl cyclase by the mutant ETA was unaffected by the mutation. In contrast, the mutated non-palmitoylated receptors but not the wild type receptor failed to stimulate phosphatidylinositol hydrolysis by phospholipase C activation upon challenge by endothelin-1. Furthermore, the mutant receptors failed to stimulate the ligand-induced transient increase in the cytoplasmic calcium seen with the wild type ETA. Endothelin-1 induced mitogenic stimuli via the wild type receptors but not through the mutated receptors suggesting an important role for phospholipase C in this signal transduction pathway. The differential regulation of distinct signal transduction pathways by post-translational modification suggests that palmitoylation of the ETA provides a novel mechanism of modulating ETA receptor activity.

Subtype-specific endothelin-A and endothelin-B receptor desensitization correlates with differential receptor phosphorylation.

In the rat cardiovascular system endothelin-1 (ET-1) elicits prolonged physiologic responses mediated by the ETA receptor, whereas the effects mediated by the ETB receptor are transient. The molecular mechanisms for the subtype-specific responses are not yet clear. However, post-translational modifications such as phosphorylation and palmitoylation may play an important role. In Sf9 cells overexpressing the human ETA and ETB receptors, both subtypes are palmitoylated. However, only the ETB but not the ETA receptor is phosphorylated in a ligand-dependent manner. Because phosphorylation is believed to play an important role in ligand-dependent receptor inactivation, we analyzed whether the differential phosphorylation of the ETA and ETB receptors reflects a differential mechanism of receptor inactivation. Using a modified inositol phosphate accumulation assay, we analyzed CHO cells that expressed the ETA or ETB receptor. The ETB receptor was deactivated almost completely within 5 min after agonist stimulation, whereas stimulation of the ETA receptor resulted in sustained activation, i.e., > 90% of the initial activity was maintained after 5 min of ligand stimulation and > 30% after 20 min. A strong correlation was observed between the time course of ETA receptor inactivation and ETA receptor internalization. The endogenous ETA receptor in Rat-1 cells produced a prolonged stimulation of phospholipase C similar to that seen in CHO cells. Therefore, the sustained signaling activity of the ETA receptor is not a property only of recombinant cell lines. Together, our data suggest rapid ETB receptor inactivation due to phosphorylation and delayed ETA receptor inactivation by internalization. These mechanisms adequately reflect the differential response patterns of the ET receptors under physiologic conditions.