Several variants of the serotonin 5-HT 4 receptor are known to be produced by alternative splicing. To survey the existence and usage of exons in humans, we cloned the human 5-HT 4 gene. Based on sequence analysis seven C-terminal variants (a-g) and one internal splice variant (h) were found. We concentrated in this study on the functional characterization of the novel splice variant h, which leads to the insertion of 14 amino acids into the second extracellular loop of the receptor. The h variant was cloned as a splice combination with the C-terminal b variant; therefore, we call this receptor 5-HT 4(hb) . This novel receptor variant was expressed transiently in COS-7 cells, and its pharmacological profile was compared with those of the previously cloned 5-HT 4(a) and 5-HT 4(b) isoforms, with the latter being the primary reference for the h variant. In competition binding experiments using reference 5-HT 4 ligands, no significant differences were detected. However, the broadly used 5-HT 4 antagonist GR113808 discriminated functionally among the receptor variants investigated. As expected, it was an antagonist on the 5-HT 4(a) and 5-HT 4(b) variant but showed partial agonistic activity on the 5-HT 4(hb) variant. These data emphasize the importance of variations introduced by splicing for receptor pharmacology and may help in the understanding of conflicting results seen with 5-HT 4 ligands in different model systems. Key Words: Serotonin 5-HT 4 receptor-Serotonin 5-HT 4(hb) variantSplice variant-G protein-coupled receptor-Gene structure -Human-Serotonin.

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.

Exogenous or endogenous injuries of the central nervous system trigger astrogliosis characterized by proliferation of astrocytes and changes in their morphology from stellate to flat polygonal. Astrocytes in culture are very sensitive to thrombin, a serine protease, which through its proteolytically activated receptor (PAR-1) induces proliferation and morphological changes comparable to astrogliosis. Evaluation of the thrombin signal-transduction pathway in the reversal of astrocyte stellation might help to understand astrogliosis. For this purpose, primary cultured murine cortical astrocytes were treated with H7, a proteinkinase inhibitor, and thrombin, which resulted in an inhibition of stellation reversal. Treatments with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, mimicked the action of thrombin. Subsequently, direct assay of astrocyte PKC activity after thrombin or PMA treatment demonstrated involvement of PKC in thrombin signaling associated with shape change. Western blotting showed that PKC isoform β-1 was involved in this pathway, while PKC A was only weakly activated and PKC β-2 was not activated by thrombin. PKC β-1 translocation was also elicited by a thrombin-receptor active peptide (SFLLRN), demonstrating the involvement of PAR-1 in this process. PKC δ and ε were located constitutively in the membrane fraction in stellate astrocytes. Isoforms γ, η, θ, and ζ were absent from astrocytes. These results suggest that astrogliosis in vivo might be regulated by modulating the activity of thrombin, PAR-1, or specific PKC isoforms.

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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Because injury of the CNS causes an astrogliosis, characterized by cell swelling and proliferation, similar to the effects of the serine protease thrombin on astrocytes, we hypothesized that a high level of thrombin at the site of injury might initially induce an astrocyte reaction and later increase the expression of its specific inhibitor, thrombomodulin. Thrombomodulin could then stabilize the astroglial scar through its adhesive properties.Here, we studied the in vivo injury response of astrocytes in the anterior medullary velum of adult rat by immunostaining and in situ hybridization of thrombomodulin. Thrombomodulin was poorly expressed on astrocytes in normal tissue, increased up to 2 d after injury, and was still highly expressed at 6 d.To check that thrombin had a direct effect on thrombomodulin expression by astrocytes, we used brain cortical astrocyte primary cultures treated with either thrombin or the agonist peptide thrombin receptor-activating peptide-6, known to activate directly the thrombin G-protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR-1). Modification of thrombomodulin expression was studied by Western blotting and quantitative reverse transcription-PCR. There was a dosedependent increase in thrombomodulin after 48 hr of treatment, with gene expression peaking at 24 hr but falling to control levels by 48 hr.Together, these results show the following: (1) injury increases astrocyte thrombomodulin expression; (2) thrombin might mediate thrombomodulin expression via the specific receptor PAR-1; and (3) serine proteases, their inhibitors, and the new family of GPCR, PARs, are active on astrogliosis.

Thrombin's potent effects on astrocytes are mediated by a specific receptor and inhibited by a serpin, protease nexin I (PNI). Thrombomodulin (TM), a membrane protein that forms complexes with thrombin, changing its enzymatic specificity, has not been studied in astrocytes. In primary astrocyte cultures, using Western blotting and immunocytochemistry, we found a 70 kDa TM band and TM localized to the surface with an anti-mouse TM monoclonal antibody. By reverse transcriptase coupled with polymerase chain reaction (RT-PCR), we found the correct sequence for mouse TM mRNA in astrocytes. Finally, we documented calcium-dependent activation of protein C by a thrombin:TM complex with thrombin added to the astrocytes. These results indicate the presence of functionally active TM at the astrocyte surface and add support to a role for thrombin signaling in the nervous system.