Various members of the canonical family of transient receptor potential channels (TRPCs) exhibit increased cation influx following receptor stimulation or Ca 2؉ store depletion. Tyrosine phosphorylation of TRP family members also results in increased channel activity; however, the link between the two events is unclear. We report that two tyrosine residues in the C terminus of human TRPC4 (hTRPC4), Tyr-959 and Tyr-972, are phosphorylated following epidermal growth factor (EGF) receptor stimulation of COS-7 cells. This phosphorylation was mediated by Src family tyrosine kinases (STKs), with Fyn appearing to be the dominant kinase. In addition, EGF receptor stimulation induced the exocytotic insertion of hTRPC4 into the plasma membrane dependent on the activity of STKs and was accompanied by a phosphorylation-dependent increase in the association of hTRPC4 with Na ؉ /H ؉ exchanger regulatory factor. Furthermore, this translocation and association was defective upon mutation of Tyr-959 and Tyr-972 to phenylalanine. Significantly, inhibition of STKs was concomitant with a reduction in Ca 2؉ influx in both native COS-7 cells and hTRPC4-expressing HEK293 cells, with cells expressing the Y959F/Y972F mutant exhibiting a reduced EGF response. These findings represent the first demonstration of a mechanism for phosphorylation to modulate TRPC channel function.The precise regulation of Ca 2ϩ signaling is crucial to the function and survival of all cell types, with disrupted Ca 2ϩ handling contributing to the pathogenesis of many diseases, including heart failure, ischemia, and neuronal degeneration (1-3). Control is maintained through complex interplays between plasma membrane proteins, the mitochondria, endoplasmic reticulum, Ca 2ϩ -binding proteins, and second messenger systems (4, 5). A ubiquitous mechanism of Ca 2ϩ entry is through the activation of non-voltage-gated Ca 2ϩ channels located in the plasma membrane. These channels respond to either receptor activation or intracellular Ca 2ϩ store depletion to control events such as gene expression, secretion, and migration (6). Various members of the transient receptor potential (TRP) 2 family of cation channels are implicated as important components of this ubiquitous Ca 2ϩ signaling pathway and represent an important area for potential drug therapy (7).The canonical or classic transient receptor potential ion channel subfamily (TRPC) is one of three TRP subfamilies known to mediate receptor-operated and store-operated Ca 2ϩ entry (7). The most compelling experimental evidence for a role of a TRPC protein in store-operated Ca 2ϩ entry came from studies on the TRPC4 null mouse where TRPC4 was identified as an essential component of store-operated Ca 2ϩ channels in aortic endothelial cells (8). Evidence for a role of TRPC channels in receptor-operated Ca 2ϩ entry include the finding that isolated endothelial cells from the TRPC4 knock-out mouse exhibited reduced Ca 2ϩ entry in response to acetylcholine, ATP, and thrombin application, and antisense oligonucleotide down-re...

Clustering of the T cell integrin, LFA-1, at specialized regions of intercellular contact initiates integrin-mediated adhesion and downstream signaling, events that are necessary for a successful immunological response. But how clustering is achieved and sustained is not known. Here we establish that an LFA-1-associated molecule, PTA-1, is localized to membrane rafts and binds the carboxyl-terminal domain of isoforms of the actinbinding protein 4.1G. Protein 4.1 is known to associate with the membrane-associated guanylate kinase homologue, human discs large. We show that the carboxylterminal peptide of PTA-1 also can bind human discs large and that the presence or absence of this peptide greatly influences binding between PTA-1 and different isoforms of 4.1G. T cell stimulation with phorbol ester or PTA-1 cross-linking induces PTA-1 and 4.1G to associate tightly with the cytoskeleton, and the PTA-1 from such activated cells now can bind to the amino-terminal region of 4.1G. We propose that these dynamic associations provide the structural basis for a regulated molecular adhesive complex that serves to cluster and transport LFA-1 and associated molecules.

T lineage-specific activation antigen 1 (TLiSA1) antigen was initially described as a T lineage-specific activation antigen involved in the differentiation of human cytotoxic T cells. Subsequently, the antigen was identified on platelets and was shown to be involved in platelet activation, hence it was renamed platelet and T cell antigen 1 (PTA1), although identity between the two antigens was not established. In the present study we have cloned the cDNA encoding TLiSA1 from Jurkat cells and show it to be a novel member of the immunoglobulin superfamily with the unusual structure of two V domains only. Identity between TLiSA1 and platelet PTA1 is established by immunological criteria, by internal peptide sequences obtained from the purified platelet glycoprotein and by sequencing the platelet transcript after reverse transcriptase-polymerase chain reaction. In Jurkat cells, TLiSA1/PTA1 mRNA and surface protein expression is greatly stimulated by treatment of the cells with phorbol ester, but the T cell proliferative signal of phorbol ester and ionophore combined greatly reduces or abrogates this response, and this suppressive effect of the ionophore is not reversed by incorporating FK506 to inhibit calcineurin. Together with the known signaling role of PTA1, these data substantiate the notion that this molecule is implicated in T cell differentiation, perhaps by engagement of an adhesive ligand.

Despite over a decade of research, only recently have the mechanisms governing transient receptor potential channel (TRPC) channel function begun to emerge, with an essential role for accessory proteins in this process. We previously identified a tyrosine phosphorylation event as critical in the plasma membrane translocation and activation of hTRPC4 channels following epidermal growth factor (EGF) receptor activation. To further characterize the signaling events underlying this process, a yeast-two hybrid screen was performed on the C terminus of hTRPC4. The intracellular C-terminal region from proline 686 to leucine 977 was used to screen a human brain cDNA library. Two members of the spectrin family, ␣II-and ␤V-spectrin, were identified as binding partners. The interaction of hTRPC4 with ␣II-spectrin and ␤V-spectrin was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation experiments. Deletion analysis identified amino acids 730 -758 of hTRPC4 as critical for the interaction with this region located within a coiled-coil domain, juxtaposing the Ca 2؉ /calmodulin-and IP 3 R-binding region (CIRB domain). This region is deleted in the proposed ␦hTRPC4 splice variant form, which failed to undergo both EGF-induced membrane insertion and activation, providing a genetic mechanism for regulating channel activity. We also demonstrate that the exocytotic insertion and activation of hTRPC4 following EGF application is accompanied by dissociation from ␣II-spectrin. Furthermore, depletion of ␣II-spectrin by small interference RNA reduces the basal surface expression of ␣hTRPC4 and prevents the enhanced membrane insertion in response to EGF application. Importantly, depletion of ␣II-spectrin did not affect the expression of the ␦ variant. Taken together, these results demonstrate that a direct interaction between hTRPC4 and the spectrin cytoskeleton is involved in the regulation of hTRPC4 surface expression and activation.

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Human T cells require two discrete signals to initiate their proliferation. In Jurkat T cells the first signal can be provided by the phorbol ester TPA and the second by the calcium ionophore A23187. We have isolated a cDNA from Jurkat T cells representing mRNA induced by TPA but inhibited by simultaneous treatment of the cells with antibody, lectin, or A23187. Sequencing revealed identity of the Jurkat clone to a cDNA, termed ETR101, recently isolated from HL60 promyelocytic leukaemia cells and shown to be an immediate early gene expressed upon TPA stimulation of these cells [Shimizu et al.: J Biol Chem 266:12157, 1991]. The gene is also induced very rapidly upon TPA treatment of Jurkat cells and is superinduced by co-treatment with cycloheximide. The predicted amino acid sequence encoded by ETR101 has weak homology to JunB and JunD, therefore it is of some interest that these three genes share the chromosomal localization, 19p13.2. The divergent effects of TPA treatment upon cell proliferation and differentiation in different circumstances allow some speculation about a possible role for the ETR101 gene product upon cellular differentiation.

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Traditional methods of dietary assessment are prone to measurement error, with energy intake often under-reported. The 24-h recall is widely used in dietary assessment, however, its reliance on self-report without verification of consumption can result in inaccuracies in true nutrient intake. Wearable cameras may provide a complementary approach to improve self-report accuracy by providing an objective and passive measure of food consumption. The purpose of the present study was to determine whether a wearable camera improves the accuracy of a 24-h recall compared with a 24-h recall alone in twenty adults aged 18–65 years. The study also explored limitations associated with wearable cameras. Participants wore the camera for 1 d and a 24-h recall was then conducted the following day, before and after viewing the camera images. Dietary data were analysed using Nutritics dietary analysis software, while eating habits were assessed by a self-report questionnaire. Energy and nutrient intakes were compared between the recall alone and the camera-assisted recall. Results showed a significant increase in mean energy intake with the camera-assisted recall compared with the recall alone (9677⋅8 ± 2708⋅0 kJ/d v. 9304⋅6 ± 2588⋅5 kJ/d, respectively, P = 0⋅003). Intakes of carbohydrates, total sugars and saturated fats were also significantly higher with the camera-assisted recall. In terms of challenges, there were occasionally technological issues such as proper positioning of the camera by the participants. In conclusion, reporting of energy and nutrient intake may be enhanced when a traditional method of dietary assessment, the 24-h recall, is assisted by a wearable camera.