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.

Previous studies on the regulation of a Ucp minigene in transgenic mice demonstrated that the sequences necessary for brown-fat-specific expression and inducibility by norepinephrine were located in the 5' flanking region between 1 and 2.8 kb from the transcriptional start site. We have investigated this region in more detail in cultured mouse brown adipocyte tumor cells. Deletion analysis of two types of chloramphenicol acetyltransferase reporter gene constructs under control of either the Ucp promoter or a heterologous herpes simplex virus-ak promoter defined an enhancer in a 220-bp HindIII-XbaI fragment which was essential for both brown fat specificity and norepinephrine inducibility. Site

Integrins play a role in the resistance of advanced cancers to radiotherapy and chemotherapy. In this study, we show that high expression of the a5 integrin subunit compromises temozolomide-induced tumor suppressor p53 activity in human glioblastoma cells. We found that depletion of the a5 integrin subunit increased p53 activity and temozolomide sensitivity. However, when cells were treated with the p53 activator nutlin-3a, the protective effect of a5 integrin on p53 activation and cell survival was lost. In a functional p53 background, nutlin-3a downregulated the a5 integrin subunit, thereby increasing the cytotoxic effect of temozolomide. Clinically, a5b1 integrin expression was associated with a more aggressive phenotype in brain tumors, and high a5 integrin gene expression was associated with decreased survival of patients with high-grade glioma. Taken together, our findings indicate that negative cross-talk between a5b1 integrin and p53 supports glioma resistance to temozolomide, providing preclinical proof-of-concept that a5b1 integrin represents a therapeutic target for high-grade brain tumors. Direct activation of p53 may remain a therapeutic option in the subset of patients with high-grade gliomas that express both functional p53 and a high level of a5b1 integrin. Cancer Res; 72(14); 3463-70. Ó2012 AACR.