Abstract
TRPV4 is a cation channel that responds to a variety of stimuli including mechanical forces, temperature, and ligand binding. We set out to identify TRPV4-interacting proteins by performing yeast two-hybrid screens, and we isolated with the avian TRPV4 amino terminus the chicken orthologues of mammalian PACSINs 1 and 3. The PACSINs are a protein family consisting of three members that have been implicated in synaptic vesicular membrane trafficking and regulation of dynamin-mediated endocytotic processes. In biochemical interaction assays we found that all three murine PACSIN isoforms can bind to the amino terminus of rodent TRPV4. No member of the PACSIN protein family was able to biochemically interact with TRPV1 and TRPV2. Co-expression of PACSIN 3, but not PACSINs 1 and 2, shifted the ratio of plasma membrane-associated versus cytosolic TRPV4 toward an apparent increase of plasma membrane-associated TRPV4 protein. A similar shift was also observable when we blocked dynamin-mediated endocytotic processes, suggesting that PACSIN 3 specifically affects the endocytosis of TRPV4, thereby modulating the subcellular localization of the ion channel. Mutational analysis shows that the interaction of the two proteins requires both a TRPV4-specific proline-rich domain upstream of the ankyrin repeats of the channel and the carboxyl-terminal Src homology 3 domain of PACSIN 3. Such a functional interaction could be important in cell types that show distribution of both proteins to the same subcellular regions such as renal tubule cells where the proteins are associated with the luminal plasma membrane.
Highlights
Human Frontier Science Program Research Grant RPG0032/2004
We provide evidence that this interaction happens between the carboxyl-terminal SH33 domain that is present in all three PACSINs and a triple-proline motif within the amino-terminal proline-rich domain (PRD) of TRPV4
Our results suggest that the mechanism of the interaction between PACSIN 3 and TRPV4 is similar to the effect of the dynamin inhibitory peptide, which supports the conclusion that PACSIN 3 inhibits TRPV4 endocytosis
Summary
Human Frontier Science Program Research Grant RPG0032/2004 The distribution of TRPV4 protein in other tissues such as airway smooth muscle, oviduct, spleen, heart, liver, testis, keratinocyte, inner ear hair cells, and dorsal root ganglion suggests that the role of this channel is not at all restricted to osmosensation This notion is supported by reports of a variety of other stimuli that activate TRPV4. A second messenger-based activation of TRPV4 is consistent with previous electrophysiological findings showing that, by switching from a cell-attached to a cell-detached patch clamp modus, the open probability of TRPV4 substantially decreases [2] This phenomenon could be interpreted as a direct result of the essential requirement of the ion channel for a specific association with intracellular structures. We report that co-expression of PACSIN 3, but not PACSINs 1 and 2, shifts the ratio between intracellular and plasma membrane-associated TRPV4 toward an apparent retention of the protein in or near the plasma membrane
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