Membrane Targeting and Coupling of NHE1-IntegrinαIIbβ3-NCX1 by Lipid Rafts following Integrin-Ligand Interactions Trigger Ca2+ Oscillations

Abstract

The cyclic calcium release and uptake during calcium oscillation are thought to result from calcium-induced calcium release (CICR); however, it is unclear, especially in nonexcitable cells, how the initial calcium mobilization that triggers CICR occurs. We report here a novel mechanism, other than conventional calcium channels or the phopholipase C-inositol trisphosphate system, for initiating calcium oscillation downstream of integrin signaling. Upon integrin ␣ IIb ␤ 3 binding to fibrinogen ligand or the disintegrin rhodostomin, sodium-proton exchanger NHE1 and sodiumcalcium exchanger NCX1 are actively transported to the plasma membrane, and they become physically coupled to integrin ␣ IIb ␤ 3 . Lipid raft-dependent mechanisms modulate the membrane targeting and formation of the NHE1-integrin ␣ IIb ␤ 3 -NCX1 protein complex. NHE1 and NCX1 within such protein complex are functionally coupled, such that a local increase of sodium concentration caused by NHE1 can drive NCX1 to generate sodium efflux in exchange for calcium influx. The resulting calcium increase inside the cell can then trigger CICR as a prelude to calcium oscillation downstream of integrin ␣ IIb ␤ 3 signaling. Fluorescence resonance energy transfer based on fluorescence lifetime measurements is employed here to monitor the intermolecular interactions among NHE1-integrin ␣ IIb ␤ 3 -NCX1, which could not be properly detected using conventional biochemical assays.In many excitable or nonexcitable cells, the concentration of free intracellular calcium oscillates with a period ranging from a few seconds to a few minutes. Such calcium oscillations are involved in a wide variety of cellular functions (1, 2). It is generally believed that, except for minor variations, the cyclic increase and decrease of calcium results from an autocatalytic release of calcium in a process called calcium-induced calcium release (CICR), 2 followed by a slow negative feedback that terminates calcium release. The cytoplasmic free calcium is then taken up into the organelles to reset the cycle. Despite a general agreement on how calcium oscillation proceeds once the system has been turned on, various different mechanisms have been proposed to explain how the initial calcium mobilization is generated that triggers CICR.As a general rule, calcium entry through voltage-gated channels in electrically excitable cells (3) or through agonist-receptor interactions in nonexcitable cells, such as epithelial cells, hepatocytes, or oocytes (4), is thought to initiate the CICR process (1, 2). Typically, in nonexcitable cells, the binding of an agonist, such as a hormone, a growth factor, or an extracellular matrix, to the corresponding cell surface receptor initiates a series of reactions that end in the activation of phopholipase C (PLC) and the production of the secondary messenger inositol trisphosphate (IP 3 ) (1, 2, 4). IP 3 is thought to induce calcium release from the internal endoplasmic reticulum or mitochondria store, and governs the CICR mechanisms that modulate calcium oscillat...

Na+/H+ Exchanger 1 Directly Binds to Calcineurin A and Activates Downstream NFAT Signaling, Leading to Cardiomyocyte Hypertrophy

“…NHE1 accumulated in restricted patches (ϳ200 nm) in the plasma membrane, observed by confocal microscopy. We observed colocalization of NHE1 with GM1 and cosedimentation of NHE1 with flotillin-1, suggesting that a substantial portion of NHE1 is localized in lipid rafts, consistent with earlier studies (5,55). Lipid rafts are dynamic assemblies enriched in sphingolipid, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins, and clustered lipid rafts may serve as heterogeneous platforms that facilitate efficient signaling (32).…”

Exposure to Host Ligands Correlates with Colocalization of Gal/GalNAc Lectin Subunits in Lipid Rafts and Phosphatidylinositol (4,5)-Bisphosphate Signaling in Entamoeba histolytica

“…It is currently unknown whether the increased intracellular calcium levels are directly related to PIP 2 hydrolysis in the cell or are attributable to other mechanisms related to calcium influx. In mammalian cells, the physical interaction between ␣ II ␤ 3 integrin, sodium-proton exchangers, and sodium-calcium exchangers occurs simultaneously with integrin binding to ligand and results in increased intracellular calcium levels (56). Additionally, in phagocytes, extracellular calcium influx was shown to be essential for movement of an integrin bound to adenylate cyclase toxin from Bordetella into lipid rafts (3).…”

Integrin-mediated Membrane Blebbing Is Dependent on Sodium-Proton Exchanger 1 and Sodium-Calcium Exchanger 1 Activity

“…Previously, we demonstrated that activation of platelet and CHO ␣IIb␤3 cells (recombinant CHO cells expressing the human integrin ␣IIb␤3 protein) by fibrinogen and recombinant rhodostomin (RGD-containing protein from Agkistrodon rhodostoma snake venom), can induce cell adhesion, spreading, and intracellular calcium oscillation (10 -14). We also reported that integrin ␣IIb␤3 downstream signals induced an interaction of NHE1-integrin ␣IIb␤3 -NCX1 on intracellular vesicles then targeting to the plasma membrane, leading to the formation of functional complexes in lipid raft microdomains (15,16). NHE1 and NCX1 both appear in homodimeric forms and are functionally coupled; NHE1 drives sodium ion influx, which in turn activates NCX1 in a reverse mode to generate a calcium influx and modulate intracellular calcium oscillations (15,(17)(18)(19)(20).…”