This study documents differences in ligand binding and signal transduction properties between the human (h) 5-hydroxytryptamine (5-HT) 4a and h5-HT 4b receptor splice variants stably expressed in human embryonic kidney 293 cells. The fraction of the [ 3 H]5-HT high-affinity site relative to the whole receptor population measured with [ 3 H]GR113808 was higher for the h5-HT 4a isoform (around 0.4) than for the 5-HT 4b isoform (around 0.2) and was independent of the level of expression. The potency and efficacy of reference compounds tested for the cAMP response differed slightly but significantly between both variants. Most remarkably, 5-methoxytryptamine and prucalopride were found more potent on the 5-HT 4b variant, whereas SDZ-HTF 919 and SB204070 were more potent on the 5-HT 4a variant. Guanosine-5Ј-O-(3-[35 S]thio)triphosphate binding on membranes and cAMP assays in whole cells revealed that only the h5-HT 4b isoform coupled to G␣i/o-proteins in addition to its well-documented G␣s coupling. In contrast, the h5-HT 4a receptor coupled only to G␣s-proteins, however, was able to trigger an increase in the intracellular calcium concentration ([Ca 2ϩ ] i ). The observed [Ca 2ϩ ] i increase did not occur through inositol phosphate formation and was not sensitive to Bordatella pertussis toxin, forskolin, or 3-isobutyl-1-methylxanthine (pre)treatment but was due to Ca 2ϩ influx from the extracellular environment. Interestingly, the Ca 2ϩ pathway was dependent on high receptor expression levels and was compound-specific, because benzamide-like compounds triggered two to three times higher responses than indoleamines. Taken together, these data provide the first evidence for fine functional differences between C-terminal splice variants of the h5-HT 4 receptor, which may contribute to a better understanding of the functional diversity of this receptor class.The ubiquitous neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) has so far been shown to interact with seven receptor classes, classified as 5-HT 1 to 5-HT 7 receptors.

The family of 5-HT4 receptors comprises 16 putative splice variants. We have previously shown that there are differences in signal transduction of the h5-HT(4a) and h5-HT(4b) receptors. In the present study, the internalization of these two splice variants following receptor stimulation was investigated with confocal microscopy on living cells. Chimeric receptors, h5-HT(4a)-GFP and h5-HT(4b)-GFP were generated by fusing the coding sequence of the 5-HT4 receptor with the coding sequence of the GFP. The agonist stimulation of fluorescent receptors resulted in a time-dependent internalization of the h5-HT(4b)-GFP receptor, but not of the h5-HT(4a)-GFP receptor. The h5-HT(4b) receptor displays a dual coupling to G(alpha)i,o and G(alpha)s proteins, in contrast to the h5-HT4a receptor, which couples to Galphas proteins only. We investigated whether the difference in internalization of the two splice variant receptors was related to their differential coupling. Therefore, we performed agonist-stimulation of the receptor following inhibition of the G(alpha)i,o protein coupling using PTX. The h5-HT(4b) receptor internalization is PTX insensitive. We co-transfected the fluorescent chimeric receptors with other wild-type variants, which did not produce an alteration of the receptor trafficking. These findings provide the first evidence of differential internalization between the two splice variants, 5-HT(4a) and 5-HT(4b) receptors.

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Fatty Acid Synthase (FASN) catalyzes the final step in palmitate (PA) synthesis, using acetyl-CoA, malonyl-CoA and NADPH. Most normal tissues express low levels of FASN and rely on uptake of fatty acids (FA) from the diet. It has been proposed that FASN overexpressing tumors including prostate and breast tumors depend on de novo FA synthesis, which is advantageous to tumors by providing lipids for membrane synthesis and increased growth factor receptor expression/signaling in lipid rafts. Overexpression of FASN leads to a higher amount of saturated lipids in membranes which can lead to resistance to cytotoxic chemotherapy. Lastly, NADPH consumption during PA synthesis keeps the redox balance in check. All of the above imply that FASN represents a potential therapeutic target for the treatment of multiple cancer types. At this AACR we report two novel chemical series (posters Connolly et al., Lu et al.). JNJ-53793220 and JNJ-54302833 potently and selectively (< 100nM) inhibit the FASN enzyme and proliferative activity in cells in lipid reduced medium (LRM). Using these, we investigated the underlying hypothesis that tumor cells do not utilize circulating FA and are dependent on de novo synthesis of FA. In a lipid reduced environment many cell lines, particularly of prostate, breast, ovarian or heme origin, proved to be sensitive to JNJ-53793220. However co-administration of PA dose-dependently reversed the anti-proliferative effects. Also androgen driven proliferation of LNCaP cells was potently blocked by JNJ-53793220 (EC50 30 nM), and decreased PSA levels. Both effects were partially rescued by the addition of PA. While the rescue of tumor cells by PA confirmed the on-target activity of the compounds, it also suggested that cancer cells are capable of using external FA. To extend these findings, we screened more than 400 cell lines in lipid containing medium (LCM) for their sensitivity to JNJ-53793220. In LCM sensitivity to FASN inhibition was lower than in LRM conditions. In most, but not all, cases the addition of PA reverted the antiproliferative effects of JNJ-53793220, although target engagement was not reduced in LCM conditions. The EC50 of 14C-acetate incorporation in lipids of ∼30 nM corresponded well with enzymatic and anti-proliferative effects in LRM (27 and 13 nM respectively). Furthermore, growth of pre-established LNCaP xenografts in vivo was not blocked significantly by JNJ-53793220, even though malonyl-CoA levels were increased as expected upon FASN inhibition in the tumor. While circulating lipids in vivo are likely culprits for the lack of efficacy, other factors may play a role as well. In a 3D culture model (poster Vidic et al.) the growth of LNCaP and PC346c spheroids was blocked by JNJ-54302833 (1µM), but growth of PC346c spheroids co-cultured with cancer associated fibroblasts was not inhibited. Taken together our data suggest that the outcome of FASN inhibition is influenced by the tumor environment. Citation Format: Karine A. Smans, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Ralph Graeser, Boudewijn Janssen, Lieven Meerpoel, Danielle Peeters, Geert Van Hecke, Luc Van Nuffel, Yolanda Chong, Peter Vermeulen, Gilles Bignan, James Bischoff, Peter Connolly, Bruce Grasberger, Tianbao Lu, Donald Ludovici, Carsten Schubert, Michael Parker, Christophe Meyer, Suzana Vidic. Sensitivity of cell lines to Fatty Acid Synthase inhibitors depends on the lipid content in the cellular environment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 801. doi:10.1158/1538-7445.AM2014-801