Mouse Trp4 and its associated proteins

Store operated channels (SOC), or also capacitative calcium entry (CCE) channels play an important role in Ca2+ homeostasis and signaling. The Drosophila transient receptor potential (Trp) gene and Trp-like (Trpl) genes encode ion channels that are activated by thapsigargin and InsP3, respectively. Mammalian homologues of the Trp gene have been proposed to be candidates for SOCs that participate in the refilling of intracellular Ca2+ stores in mammalian cells. Although the stoichiometry and pharmacology of these newly cloned channels have not been elucidated, they provided a basis on which to study how the state of intracellular calcium stores is communicated to the channels on the plasma membrane. Here we report the cloning and expression of mouse Trp4 and a splice variant mTrp4S (84 amino acid deletion) from a mouse insulinoma cDNA library, along with four cDNAs identified by their ability to bind to mTrp4 in the yeast two-hybrid system. RNA blotting studies showed that mTrp4 is highly expressed in brain and in endocrine cells but not in spleen or liver. mTrp4 encoded a protein of 97 KD in a coupled transcription-translation in vitro system. Stable cell lines expressing mTrp4, and tagged Trp4 fusion proteins, showed abundant intracellular Trp4 expression detected with antibodies directed against either a C-terminal peptide or the epitope tag. Cell lines expressing hTrp3 but not mTrp4 alone, showed Gq protein-coupled receptor activation of calcium influx. Four candidate Trp4 binding proteins, termed Trp-associated proteins, or Taps were identified using the yeast two-hybrid system. We further characterized Tap1 in detail. Mouse Tap1, which encodes a novel protein of 79 KD, is widely expressed in mammalian tissues, and is part of an unreported gene family highly conserved in vertebrates. Two Tap1 homologues were obtained from Danio rerio (zTap1A and zTap1B, 74% and 69% identify to mTap1 respectively) and were found to have a ubiquitous pattern of expression in Danio rerio embryos as well. These studies further our understanding of the complexities of Trp gene expression and function and suggest that multiple novel proteins may participate in mammalian Trp function or trafficking.