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Functional role of C-terminal cytoplasmic tail of rat vanilloid receptor 1.

ABSTRACT: The vanilloid receptor [transient receptor potential (TRP)V1, also known as VR1] is a member of the TRP channel family. These receptors share a significant sequence homology, a similar predicted structure with six transmembrane-spanning domains (S1-S6), a pore-forming region between S5 and S6, and the cytoplasmically oriented C- and N-terminal regions. Although structural/functional studies have identified some of the key amino acids influencing the gating of the TRPV1 ion channel, the possible contributions of terminal regions to vanilloid receptor function remain elusive. In the present study, C-terminal truncations of rat TRPV1 have been constructed to characterize the contribution of the cytoplasmic C-terminal region to TRPV1 function and to delineate the minimum amount of C tail necessary to form a functional channel. The truncation of 31 residues was sufficient to induce changes in functional properties of TRPV1 channel. More pronounced effects of C-terminal truncation were seen in mutants lacking the final 72 aa. These changes were characterized by a decline of capsaicin-, pH-, and heat-sensitivity; progressive reduction of the activation thermal threshold (from 41.5 to 28.6 degrees C); and slowing of the activation rate of heat-evoked membrane currents (Q10 from 25.6 to 4.7). The voltage-induced currents of the truncated mutants exhibited a slower onset, markedly reduced outward rectification, and significantly smaller peak tail current amplitudes. Truncation of the entire TRPV1 C-terminal domain (155 residues) resulted in a nonfunctional channel. These results indicate that the cytoplasmic COOH-terminal domain strongly influences the TRPV1 channel activity, and that the distal half of this structural domain confers specific thermal sensitivity.

SUBMITTER: Vlachova V

PROVIDER: S-EPMC6742269 | biostudies-literature | 2003 Feb

REPOSITORIES: biostudies-literature

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Functional role of C-terminal cytoplasmic tail of rat vanilloid receptor 1.

Vlachová Viktorie V Teisinger Jan J Susánková Klára K Lyfenko Alla A Ettrich Rüdiger R Vyklický Ladislav L

The Journal of neuroscience : the official journal of the Society for Neuroscience 20030201 4

The vanilloid receptor [transient receptor potential (TRP)V1, also known as VR1] is a member of the TRP channel family. These receptors share a significant sequence homology, a similar predicted structure with six transmembrane-spanning domains (S1-S6), a pore-forming region between S5 and S6, and the cytoplasmically oriented C- and N-terminal regions. Although structural/functional studies have identified some of the key amino acids influencing the gating of the TRPV1 ion channel, the possible ...[more]

PMID: 12598622

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Project description:The C-terminal tail of G-protein-coupled receptors (GPCR) contain important regulatory sites that enable interaction with intracellular signaling effectors. Here we examine the relative contribution of the C-tail serine/threonine phosphorylation sites (Ser383-385, Ser387-Thr392) and the helix-8 palmitoylation site (Cys361) in signaling regulation downstream of the proteolytically activated GPCR, PAR2. We examined Gαq/11-coupled calcium signaling, β-arrestin-1/-2 recruitment, and MAPK activation (p44/42 phosphorylation) by wild-type and mutant receptors expressed in a CRISPR/Cas9 PAR2-knockout HEK-293 cell background with both peptide stimulation of the receptor (SLIGRL-NH2) as well as activation with its endogenous trypsin revealed a tethered ligand. We find that alanine substitution of the membrane proximal serine residues (Ser383-385Ala) had no effect on SLIGRL-NH2- or trypsin-stimulated β-arrestin recruitment. In contrast, alanine substitutions in the Ser387-Thr392 cluster resulted in a large (∼50%) decrease in β-arrestin-1/-2 recruitment triggered by the activating peptide, SLIGRL-NH2, but was without an effect on trypsin-activated β-arrestin-1/-2 recruitment. Additionally, we find that alanine substitution of the helix-8 cysteine residue (Cys361Ala) led to a large decrease in both Gαq/11 coupling and β-arrestin-1/-2 recruitment to PAR2. Furthermore, we show that Gαq/11 inhibition with YM254890, inhibited ERK phosphorylation by PAR2 agonists, while genetic deletion of β-arrestin-1/-2 by CRISPR/Cas9 enhanced MAPK activation. Knockout of β-arrestins also enhanced Gαq/11-mediated calcium signaling. In line with these findings, a C-tail serine/threonine mutant that has decreased β-arrestin recruitment also showed enhanced ERK activation. Thus, our studies point to multiple mechanisms that regulate β-arrestin interaction with PAR2 and highlight differences in regulation of tethered-ligand- and peptide-mediated activation of this receptor.

Project description:The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Calpha (PKCalpha) to membrane was similar to that of DG formation. In WT-H cells, PKCalpha was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCalpha was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCalpha. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that sustained PLD activation in turn leads to chronic activation and membrane translocation of PKCalpha, which might play an important role in the expression of c-fos and c-jun.

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Functional role of C-terminal cytoplasmic tail of rat vanilloid receptor 1.

Publications

Functional role of C-terminal cytoplasmic tail of rat vanilloid receptor 1.

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