Translocation of Endothelial Nitric-Oxide Synthase Involves a Ternary Complex with Caveolin-1 and NOSTRIN

Abstract

Recently, we characterized a novel endothelial nitric-oxide synthase (eNOS)-interacting protein, NOSTRIN (for eNOStrafficking inducer), which decreases eNOS activity upon overexpression and induces translocation of eNOS away from the plasma membrane. Here, we show that NOSTRIN directly binds to caveolin-1, a well-established inhibitor of eNOS. Because this interaction occurs between the N terminus of caveolin (positions 1-61) and the central domain of NOSTRIN (positions 323-434), it allows for independent binding of each of the two proteins to eNOS. Consistently, we were able to demonstrate the existence of a ternary complex of NOSTRIN, eNOS, and caveolin-1 in Chinese hamster ovary (CHO)-eNOS cells. In human umbilical vein endothelial cells (HUVECs), the ternary complex assembles at the plasma membrane upon confluence or thrombin stimulation. In CHO-eNOS cells, NOSTRIN-mediated translocation of eNOS involves caveolin in a process most likely representing caveolar trafficking. Accordingly, trafficking of NOSTRIN/eNOS/ caveolin is affected by altering the state of actin filaments or cholesterol levels in the plasma membrane. During caveolar trafficking, NOSTRIN functions as an adaptor to recruit mediators such as dynamin-2 essential for membrane fission. We propose that a ternary complex between NOSTRIN, caveolin-1, and eNOS mediates translocation of eNOS, with important implications for the activity and availability of eNOS in the cell. INTRODUCTIONEndothelial nitric-oxide synthase (eNOS) is the major enzyme generating nitric oxide (NO) in endothelial and epithelial cells (Ortiz and Garvin, 2003;Sessa, 2004). Because NO is an extremely reactive signaling molecule its production needs to be tightly regulated. Regulation seems to take place at three levels: direct interaction of eNOS with accessory proteins such as caveolin and Ca 2ϩ /calmodulin, reversible phosphorylation, and differential localization of the enzyme within cells. Subcellular distribution of eNOS is in part governed by lipid modification, i.e., myristoylation and dual palmitoylation, which bring about the association of the enzyme with the Golgi and plasma membrane (PM), respectively (Govers and Rabelink, 2001). Importantly, eNOS seems to be regulated by different modes in different subcellular locations, e.g., Ca 2ϩ /calmodulin stimulation is mainly effective at the PM, whereas Akt-driven activation is most pronounced at the Golgi (Fulton et al., 2004). Differential subcellular localization of eNOS is subject to dynamic regulation, e.g., after certain stimuli, the enzyme also occurs at vesicular structures throughout the cytoplasm (Nuszkowski et al., 2001;Thuringer et al., 2002). At present, however, it remains largely unknown how the differential distribution of eNOS to various subcellular locales is achieved. Emerging determinants of eNOS trafficking are eNOS-interacting proteins, which may guide eNOS to a distinct destination within the cell. In support of this model, we described two novel eNOS-interacting proteins termed NOSIP (for eNOS-inter...

Knockdown of the Hypertension-Associated Gene NOSTRIN Alters Glomerular Barrier Function in Zebrafish ( Danio rerio )

“…15,16 More recently, the same group showed that NOSTRIN is involved in vascular development and that loss of NOSTRIN resulted in impaired vascular patterning. Here, we showed that in human and rodent glomeruli, NOSTRIN is expressed predominantly in epithelial podocytes and interacts with proteins such as CD2AP that are known to be essential for proper slit-membrane function.…”

“…15 It has been attributed to the control of endothelial nitric oxide synthase activity and has been shown to interact with dynamin-2, neural WiskottAldrich syndrome protein, and caveolin-1. 15,16 More recently, the same group showed that NOSTRIN is involved in vascular development and that loss of NOSTRIN resulted in impaired vascular patterning. 9 Here, we showed that in human and rodent glomeruli, NOSTRIN is expressed predominantly in epithelial podocytes and interacts with proteins such as CD2AP that are known to be essential for proper slit-membrane function.…”

Modulating endothelial nitric oxide synthase: a new cardiovascular therapeutic strategy

“…Two other proteins named as eNOS-interacting protein (NOSIP) and eNOS traffic inducer (NOSTRIN) that specifically modulate the caveolin/eNOS interaction have been described (33,113,165). NOSTRIN also functions as an adaptor protein to recruit dynamin-2, which is a large GTPase and a positive modulator of eNOS (118) to facilitate internalization of eNOS (113).…”

The Physiological Relationship of Endothelial Protein Kinase G with Endothelial Nitric Oxide Synthase

“…42,43 Previously, factors modulating NOS were studied largely by investigating stimulated NOS activity, NOS activity in vitro and in transfection-modified cells using mainly the citrulline assay. Such activation and the interaction of NOS and its binding partners may be modulated by proteins such as heat shock protein 90 (HSP90), 44 nitric oxide synthase traffic inducer (NOSTRIN), 45 and eNOS interacting protein (NOSIP), 46 as well as kinase action by Akt (protein kinase B, PKB) and PKA (protein kinase A). 47 Basal endothelial nitric oxide production has been difficult to measure and study with the citrulline assay.…”