AbstractAlthough most signaling responses initiated by tumor necrosis factor‐α (TNF‐α) occur in a Ca2+‐independent fashion, TNF‐α receptor signaling augments Ca2+ entry induced by Gαq/11 G‐protein coupled receptors (GPCRs) in endothelial cells and increases trans‐endothelial permeability. The signaling events involved in GPCR‐induced Ca2+ influx have been characterized and involve store‐operated Ca2+ entry facilitated by the Ca2+ permeable ion channel, transient receptor potential canonical 4 (TRPC4). Little is known about the mechanisms by which TNF‐α receptor signaling augments GPCR‐induced Ca2+ entry. TNF‐α Receptor Ubiquitous Signaling and Scaffolding protein (TRUSS) is a tumor necrosis factor receptor‐1 (TNF‐R1)‐associated protein whose gene name is TRPC4‐associated protein (TRPC4AP). The goal of our study was to test the hypothesis that TRUSS serves to link TNF‐R1 and GPCR‐signaling pathways at the level of TRPC4 by: (i) determining if TRUSS and TNF‐R1 interact with TRPC4, and (ii) investigating the role of TRUSS, TNF‐R1, and TRPC4 in GPCR‐induced Ca2+ signaling. Here, we show that TRUSS and TNF‐R1 interact with a sub‐family of TRPC channels (TRPC1, 4, and 5). In addition, we show that TRUSS and TNF‐R1 function together with TRPC4 to elevate endoplasmic reticulum Ca2+ filling in the context of reduced endoplasmic reticulum Ca2+ storage initiated by G‐protein coupled m1 muscarinic acetylcholine receptor (m1AchR) signaling. Together, these findings suggest that TNF‐R1, TRUSS, and TRPC4 augment Ca2+ loading of endoplasmic reticulum Ca2+ stores in the context of m1AchR stimulation and provide new insights into the mechanisms that connect TNF‐R1 to GPCR‐induced Ca2+ signaling. J. Cell. Physiol. 225: 444–453, 2010. © 2010 Wiley‐Liss, Inc.