Assembly of Trp1 in a Signaling Complex Associated with Caveolin-Scaffolding Lipid Raft Domains

Abstract

Trp1 has been proposed as a component of the storeoperated Ca 2؉ entry (SOC) channel. However, neither the molecular mechanism of SOC nor the role of Trp in this process is yet understood. We have examined possible molecular interactions involved in the regulation of SOC and Trp1 and report here for the first time that Trp1 is assembled in signaling complex associated with caveolin-scaffolding lipid raft domains. Endogenous hTrp1 and caveolin-1 were present in low density fractions of Triton X-100-extracted human submandibular gland cell membranes. Depletion of plasma membrane cholesterol increased Triton X-100 solubility of Trp1 and inhibited carbachol-stimulated Ca 2؉ signaling. Importantly, thapsigargin stimulated Ca 2؉ influx, but not internal Ca 2؉ release, and inositol 1,4,5-triphosphate (IP 3 )-stimulated I soc were also attenuated. Furthermore, both anti-Trp1 and anti-caveolin-1 antibodies co-immunoprecipitated hTrp1, caveolin-1, G␣ q/11 , and IP 3 receptortype 3 (IP 3 R3). These results demonstrate that caveolar microdomains provide a scaffold for (i) assembly of key Ca 2؉ signaling proteins into a complex and (ii) coordination of the molecular interactions leading to the activation of SOC. Importantly, we have shown that Trp1 is also localized in this microdomain where it interacts with one or more components of this complex, including IP 3 R3. This finding is potentially important in elucidating the physiological function of Trp.

A polycystin multiprotein complex constitutes a cholesterol-containing signalling microdomain in human kidney epithelia

“…We envision that flotillin-2 and cholesterol in the polycystin multiprotein complex similarly co-ordinate the close spatial clustering of the polycystins with the E-cadherin/β-catenin cell-cell adhesion molecules and regulatory signalling molecules. The integration of polycystin-2-related calcium channels, such as TrpC1, in caveolin membrane domains is suggested to promote close association with regulatory G-proteins and is functionally important for Ca 2+ signalling [16,42]. Interestingly, flotillin-2 overexpression has been linked to the co-ordinate overexpression of a G-protein-coupled receptor in melanoma cells [43].…”

Free Cholesterol Alters Lipid Raft Structure and Function Regulating Neutrophil Ca2+ Entry and Respiratory Burst: Correlations with Calcium Channel Raft Trafficking

“…1A). When we used M␤CD to remove cholesterol from and thus disrupt rafts (21,31,32), we found that Ca 2ϩ entry was blocked by cholesterol sequestration and small amounts of cholesterol rescued Ca 2ϩ entry. Cholesterol depletion had no effect whatsoever on release of Ca 2ϩ from cell stores, demonstrating that the afferent arm of GPCR signaling was not affected by raft disruption.…”

“…PMN lack voltage-gated calcium channels (19) and we have shown that PMN mobilize external Ca 2ϩ via mechanisms that mobilize multiple TRPC proteins to the cell membrane (8,9). Cytoskeletal alterations that prevent TRPC membrane localization block entry of calcium into PMN (9) and TRPC are known to localize to rafts in PMN and other cell types (20,21).…”

Cutting Edge: Optical Microspectrophotometry Supports the Existence of Gel Phase Lipid Rafts at the Lamellipodium of Neutrophils: Apparent Role in Calcium Signaling

“…1B). Similarly, the Ca 2ϩ channel TRPC-1, which also cofractionates with lipid rafts (14), collects at lamellipodia of polarized cells (94 Ϯ 5%) (Fig. 1F).…”