The human 5-HT 7 serotonin receptor splice variants: constitutive activity and inverse agonist effects (original) (raw)
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Naunyn-Schmiedeberg's Archives of Pharmacology, 2001
Serotonin (5-hydroxytryptamine, 5-HT) receptor pre-mRNA is alternatively spliced in human tissue to produce three splice variants, h5-HT 7(a) , h5-HT 7(b) and h5-HT 7(d) , which differ only in their carboxyl terminal tails. Using membranes from transiently and stably transfected HEK293 cells expressing the three recombinant h5-HT 7 splice variants we compared their pharmacological profiles and ability to activate adenylyl cyclase. Using PCR on cDNA derived from various human tissues, the 5-HT 7(a) and 5-HT 7(b) splice variants were detected in every tissue examined. The h5-HT 7(d) splice variant was detected in 13 of 16 tissues examined, with predominant expression in the heart, small intestine, colon, ovary and testis. All three h5-HT 7 splice variants displayed high affinity binding for [ 3 H]5-HT (pK d =8.8-8.9) in the presence and absence of 100 µM GTP and had similar binding affinities for all 17 ligands evaluated. In HEK293 cells expressing similar, high levels of receptor (~10,000 fmol/mg protein), 5-CT (5-carboxamidotryptamine), 5-MeOT (5-methoxytryptamine) and 5-HT were full agonists while 8-OH-DPAT ((2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin) was a partial agonist with relative efficacy of ~0.8. Even at this high receptor level, EC 50 values for stimulation of adenylyl cyclase were 10-to 50-fold higher than the K d values, indicating a lack of spare receptors. No significant differences in coupling to adenylyl cyclase were observed between the three splice variants over a wide range of receptor expression levels. For antagonists, binding affinities determined by displacement of [ 3 H]5-HT binding and by com-petitive inhibition of 5-HT-stimulated adenylyl cyclase activity were essentially identical amongst the splice variants. These studies indicate that the three human splice variants are pharmacologically indistinguishable and that modifications of the carboxyl tail do not influence coupling to adenylyl cyclase.
2004
5-HT 4 receptor pre-mRNA is alternatively spliced in human (h) tissue to produce several splice variants, called 5-HT 4(a) to 5-HT 4(h) and 5-HT 4(n) . Polymerase chain reaction (PCR) with primers designed to amplify both 5-HT 4(a) and 5-HT 4(b) amplified three additional bands in different tissues, two representing different mRNA species both encoding 5-HT 4(g) and one representing mRNA for a novel splice variant named 5-HT 4(i) , cloned from testis and pancreas respectively. Primary and nested PCR detected both 5-HT 4(g) and 5-HT 4(i) in multiple tissues. Whereas 5-HT 4(i) , was found in all cardiovascular tissues analysed, 5-HT 4(g) was mainly present in atria. However, quantitative RT-PCR indicated 5-HT 4(g) expression also in cardiac ventricle. The pharmacological profiles and ability to activate adenylyl cyclase (AC) were compared between four recombinant h5-HT 4 splice variants (a, b, g and i) expressed transiently and stably in HEK293 cells. Displacement of [ 3 H]GR113808 with ten ligands revealed identical pharmacological profiles (affinity rank order: GR125487, SB207710, GR113808>SB203186>serotonin, cisapride, tropisetron>renzapride, 5-MeOT>5-CT). In transiently transfected HEK293 cells cisapride was a partial agonist compared to serotonin at 5-HT 4(b) , 5-HT 4(g) and 5-HT 4(i) receptors. In membranes from HEK293 cells stably expressing 5-HT 4(g) (3,000 fmol/mg protein) or 5-HT 4(i) (500 fmol/mg protein), serotonin and 5-MeOT were full agonists while cisapride was full agonist at 5-HT 4(g) and partial agonist at 5-HT 4(i) , probably due to different receptor expression levels. At both 5-HT 4(g) and 5-HT 4(i) , the behaviour of 5-HT 4 receptor antagonists was dependent on receptor level. At high receptor levels, tropisetron and SB207710 and to a variable extent SB203186 and GR113808 displayed some partial agonist activity, whereas GR125487 and SB207266 reduced the AC activity below basal, indicating both receptors to be constitutively active. We conclude that the novel 5-HT 4(i) receptor splice variant is pharmacologically indistinguishable from other 5-HT 4 splice variants and that the 5-HT 4(i) C-terminal tail does not influence coupling to AC.
Naunyn-Schmiedeberg's Archives of Pharmacology, 1992
The characteristics of 5-HT~A-recognition sites and receptor-mediated release of intracellular calcium were established in two transfected HeLa cell lines (HA 6 and HA 7) expressing different levels of human 5-HT~A receptors (about 3000 and 500 fmol/mg protein, Fargin et al. 1989; 1991; Raymond et al. 1989). The pharmacological profiles of the binding (determined with [3H]8-OH-DPAT) and the calcium response (measured using Fura-2) were clearly of the 5-HT 1A type. Compounds such as 5-HT, 5-CT and 8-OH-DPAT acted as full agonists on the calcium response in both HeLa cell lines. In addition, methiothepin, pindolol, NAN 190 and SDZ 216-525 (Seiler et al. 1991) acted as silent and potent antagonists. Marked differences were observed in the responses mediated in the two cell lines. ECso values of agonists (particularly 5-HT, 5-CT, flesinoxan and 8-OH-DPAT) were higher in HA 7 cells (up to 80-fold) than in other 5-HT1A receptor models (e.g. inhibition of adenylate cyclase in calf hippocampus). Further, a variety of compounds (ipsapirone, buspirone, spiroxatrine, MDL 73005) acted as agonists in HA 6 cells, whereas they behaved as silent antagonists in HA 7 cells (which express fewer receptors). By contrast, KB values for antagonists were comparable in HA 6 and HA 7 cells. The present data show that ECs0 values and intrinsic activity for a given drug are subject to large variations depending on the number of receptors expressed in the target tissue. The results obtained in HA 6 cells are comparable with respect to both potency and efficacy to those observed.in calf or mouse hippocampus (inhibition of forskolin stimulated adenylate cyclase), whereas the results obtained in HA 7 cells are similar to those reported in mouse cortex (which was suggested to represent an atypical subtype of the 5-HTIA receptor). Since the agonist activity of a given compound at the same receptor can vary markedly, the present data show Send offprint requests to D. Hoyer at the above address J. R. Raymond was supported by the American Heart Association (Grant in Aid) and by a V. A. Merit Award that intrinsic activity is not only ligand-dependent but also varies with the receptor-effector system studied. In addition, there seems to be no simple way to make predictions about intrinsic activity, since that feature is modeldependent.
Proceedings of The National Academy of Sciences, 1993
An intronless gene encoding an additional human serotonin (5-1T) 5-HTificee receptor subtype was isolated from a human genomic library with probes obtained from degenerate PCR primers used to amplify 5-liT-receptorspecific sequences. The highest degree of homology was found with the 5-HT1E subtype (70%) and the 5-HTlDa (63%) and 5-HTlDp3 (60%) receptors. RNA for this gene was detected in the human brain but was not detected in kidney, liver, spleen, heart, pancreas, and testes. High-affinity (Kd = 9.2 nM) 3H-labeled 5-HT binding was detected. Competition studies revealed the following rank order of potencies for serotonergic ligands: 5-HT > sumatriptan >> 5-carboxyamidotryptamine > 8-hydroxy-2(di-1-propylamino)tetralin > spiperone. 5-HT produced a dose-dependent inhibition of forskolin-stlmulated cAMP accumulation (EC50 = 7.9 nM) in transfected cells.
Naunyn-schmiedebergs Archives of Pharmacology, 2006
The present study reinvestigated a series of 5-HT receptor antagonists at both constitutively active rat and human 5-HT7(a) receptors in HEK-293F cells using the cAMP signalling pathway as a functional read-out. Both rat and human 5-HT7(a) receptors were expressed in similar amounts ([3H]-LSD binding: 1.0 to 1.1 pmol/mg protein). Attenuation of basal cAMP formation by the inverse agonist SB-691673 (1 μM) was slightly larger by the human 5-HT7(a) (−73±3 %) than rat 5-HT7(a) receptor (−62±3 %). The 5-HT receptor antagonists investigated here displayed systematically inverse agonism. While methiothepin and SB-269970 displayed similar negative intrinsic activity to SB-691673 at the rat 5-HT7(a) receptor, the compounds SB-258719, mesulergine and metergoline displayed some lower negative intrinsic activity (between −38 and −49%). Inverse agonist properties were observed with potencies fitting with their respective binding pIC50 values and pKB values as estimated from antagonist studies with 5-HT. With the exception of SB-258719 and mesulergine, which remained a partial inverse agonist at the human 5-HT7(a) receptor, the other compounds behaved with a similar Emax value to the full inverse agonist SB-691673. In conclusion, none of the 5-HT receptor antagonists investigated displayed silent properties at the rat or human 5-HT7(a) receptor, when these are expressed in a system allowing detection of constitutive activity. They appear to be partial to full inverse agonists, further illustrating that an antagonist is preferentially an inverse agonist when investigated under constitutively active receptor conditions.
British Journal of Pharmacology, 1997
1The rat 5-hydroxytryptamine (5-HT)7 receptor displays two splice variations, a long form, and a truncated splice isoform, arising from the introduction of a stop codon near the carboxy-terminus. The human 5-HT7 receptor gene contains at least two introns and encodes a 445 amino acid 5-HT receptor.2A truncated splice variation in the human 5-HT7 receptor was isolated from a human placental cDNA library. In accordance with current NC-IUPHAR nomenclature guidelines, it is suggested that this receptor be denoted as the h5-HT7(b) receptor and the long form of the receptor as h5-HT7(a).3The h5-HT7(b) receptor was stably expressed in HEK 293 cells and ligand affinities were determined by displacement of [3H]-5-carboxyamidotryptamine (5-CT; Kd=0.28±0.06 nM, Bmax=7.3±1.7 pmol mg−1 protein). The rank order of affinities (pKi) for a series of ligands was: 5-carboxamidotryptamine (5-CT, 9.65)>5-hydroxytryptamine (5-HT, 9.41)>methiothepin (8.87)>mesulergine (7.87)>8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT, 6.85)>ketanserin (6.44).4The h5-HT7(b) receptor coupled positively to adenylyl cyclase in HEK 293 cells. This response was elicited by a number of agonists with the following order of potency (pEC50): 5-CT (8.7±0.11)>5-MeOT (5-methoxytryptamine; 8.1±0.20)>5-HT (7.5±0.13)>tryptamine (5.6±0.36)>8-OH-DPAT (5.3±0.28)>5-methoxytryptamine (5.0±0.06). This rank order was comparable to that observed in the radioligand binding studies.5In a similar fashion to that described for the 5-HT7(a) receptor, PCR studies suggested that the 5-HT7(b) receptor mRNA is found in great abundance throughout the brain, in the small intestine and aorta.6It is concluded that the h5-HT7 receptor, like the rat receptor, exists as splice variants exhibiting similar pharmacology, signal transduction and distribution. It is thus likely that there exists a complex physiological role for alternate splicing products of the 5-HT7 receptor gene.The rat 5-hydroxytryptamine (5-HT)7 receptor displays two splice variations, a long form, and a truncated splice isoform, arising from the introduction of a stop codon near the carboxy-terminus. The human 5-HT7 receptor gene contains at least two introns and encodes a 445 amino acid 5-HT receptor.A truncated splice variation in the human 5-HT7 receptor was isolated from a human placental cDNA library. In accordance with current NC-IUPHAR nomenclature guidelines, it is suggested that this receptor be denoted as the h5-HT7(b) receptor and the long form of the receptor as h5-HT7(a).The h5-HT7(b) receptor was stably expressed in HEK 293 cells and ligand affinities were determined by displacement of [3H]-5-carboxyamidotryptamine (5-CT; Kd=0.28±0.06 nM, Bmax=7.3±1.7 pmol mg−1 protein). The rank order of affinities (pKi) for a series of ligands was: 5-carboxamidotryptamine (5-CT, 9.65)>5-hydroxytryptamine (5-HT, 9.41)>methiothepin (8.87)>mesulergine (7.87)>8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT, 6.85)>ketanserin (6.44).The h5-HT7(b) receptor coupled positively to adenylyl cyclase in HEK 293 cells. This response was elicited by a number of agonists with the following order of potency (pEC50): 5-CT (8.7±0.11)>5-MeOT (5-methoxytryptamine; 8.1±0.20)>5-HT (7.5±0.13)>tryptamine (5.6±0.36)>8-OH-DPAT (5.3±0.28)>5-methoxytryptamine (5.0±0.06). This rank order was comparable to that observed in the radioligand binding studies.In a similar fashion to that described for the 5-HT7(a) receptor, PCR studies suggested that the 5-HT7(b) receptor mRNA is found in great abundance throughout the brain, in the small intestine and aorta.It is concluded that the h5-HT7 receptor, like the rat receptor, exists as splice variants exhibiting similar pharmacology, signal transduction and distribution. It is thus likely that there exists a complex physiological role for alternate splicing products of the 5-HT7 receptor gene.British Journal of Pharmacology (1997) 122, 126–132; doi:10.1038/sj.bjp.0701336
Journal of Pharmacy and Pharmacology, 2004
Twenty agonists and nine antagonists were evaluated for their ability to compete for [ 3 H]-8-hydroxy-2-(di-n-propylamino)tetralin ([ 3 H]-8-OH-DPAT) binding to the cloned human serotonin-1A (ch-5-HT 1A ) receptor expressed in Chinese hamster ovary cells and for their ability to alter adenylyl cyclase activity in the same cells. The most potent full agonists of high affinity included N,N-dipropyl-5-carboxamidotryptamine (pEC50 = 9.6 -0.1), MDL 73005EF (pEC50 = 9.3 -0.2), 5-methyl-urapidil (pEC50 = 9.2 -0.1), 5carboxamidotryptamine (pEC50 = 9.1 -0.2), R(þ)-8-OH-DPAT (pEC50 = 8.6 -0.1) and BMY-7378 (pEC50 = 8.6 -0.1). WB-4101 (pEC50 = 8.3 -0.2; IA = 79%), clozapine (pEC50 = 8.1 -0.3; IA = 29%), (buspirone (pEC50 = 7.6 -0.2; IA = 79%), quipazine (pEC50 < 5; IA = 45%) and R-DOI (pEC50 < 5; IA = 31%) were weaker agonists with partial agonist properties. The most potent antagonists were WAY-100,635 (pK i = 10.2 -0.1), methiothepin (pK i = 8.8 -0.2), spiperone (pK i = 8.7 -0.2) and NAN-190 (pK i = 8.5 -0.2). The receptor affinities and functional potencies were well correlated (r = 0.88; P < 0.0001). Our binding data correlated well with the pharmacology of endogenous 5-HT 1A receptors in the rabbit iris-ciliary body (r = 0.91; P < 0.001) and rat hippocampus (r = 0.93, P < 0.0001). Our functional cAMP data correlated well with other cAMP accumulation data (r = 0.8, P < 0.01 vs calf hippocampus) but less so with [ 35 S]-GTPS binding to the ch-5-HT 1A receptor as a functional activity read-out (r = 0.58, P < 0.05). The present study provides a detailed pharmacological characterization of the ch-5-HT 1A receptor using binding and functional assays.