Members of the canonical transient receptor potential (TRPC) family of non-selective cation channels are implicated in the regulation of both store-operated (SOC) and receptor-operated (ROC) Ca²⁺ entry (1). The assembly of TRPC channels into signalling micro-domains is important for the control of channel expression, localization and ultimately activation (2). Disruption of these complexes results in altered Ca²⁺ signalling with potentially deleterious cellular consequences (3). We have investigated the formation of protein complexes with hTRPC4 and their implications for channel function. We demonstrate biochemically, through discontinuous sucrose gradients, that hTRPC4 resides in lipid-raft domains (LRDs) enriched with caveolin-1. 81% of endogenous hTRPC4 in A431 cells (n=6) and 90% of transfected hTRPC4 resides within low-density fractions 3-5 associated with caveolin-1. By comparison, 48% of αII-spectrin (n=11) and approximately 100% of caveolin-1 (n=6) migrate within these fractions. These LRDs are important in the control of channel surface expression, with disruption dramatically increasing the level of hTRPC4 approximately 8-fold (n=5) at the plasma membrane. This internal supply of channels provides a readily releasable pool of hTRPC4 for insertion following stimulation with epidermal growth factor (EGF). We provide evidence that this membrane insertion is accompanied by the dissociation of hTRPC4 from the spectrin cytoskeleton and is driven by the phosphorylation of C-terminal tyrosine residues by Src-family non-receptor tyrosine kinases (STKs). In agreement, the STK members Fyn and Lyn were shown to co-immunoprecipitate with TRPC4 from rat brains of humanely killed rats and COS-7 cells, respectively. The phosphorylation of hTRPC4 also enhanced its association with the scaffolding protein, Na⁺/H⁺ exchanger regulatory factor, NHERF. We speculate that this association is responsible for the recruitment and stabilisation of hTRPC4 channels at the plasma membrane following EGF application. Furthermore, the phosphorylation of hTRPC4 by STKs is critical for the maintenance of EGF-stimulated Ca²⁺ entry in HEK293 cells stably expressing hTRPC4. The expression of hTRPC4 increased Ca²⁺ influx 2-fold (70.5nM, n=7) when compared to control GFP-expressing cells (35.1nM, n=5) following EGF stimulation. The application of 5μM PP2 prior to EGF stimulation prevented this increase (31.4nM, n=4). Taken together, these data demonstrate the formation of functional hTRPC4 signalling complexes consisting of channels, spectrin, NHERF, and non-receptor tyrosine kinases. These complexes reside in LRDs and are activated by growth factor receptor stimulation to initiate Ca²⁺ influx.