Cloning and Expression of a Human Serotonin 5HT4 Receptor cDNA (original) (raw)
Related papers
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
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.
Neuropharmacology, 1997
Sunun ary-RS 576391, by being a partial agonist in rat esophagusbut a competitive antagonistin guinea-pig ileum, is one of several ligands which operationally discriminate among 5-HT4receptors in different tissues. The discovery-of splice variants of the 5-HT4receptor, 5-HT4sand 5-HT4~,raises the possibility that this functional heterogeneity among 5-HT4receptors may be due to differences in the interaction of ligands with differentisoformsof the receptor.To test this idea, the functionaland bindinginteractions of RS 57639with rat 5-HT4sand 5-HTw receptorswere characterized.RS 57639stimulated adenylatecyclase in cells expressing5-HT4s or 5-HT4~receptors with similar potency (pEC~O = 7.9 + 0.1 and 7.6~0.1) and efficacy (71 + 3 and 59~4% of 5-HT).
Differential Agonist-Mediated Internalization of the Human 5-Hydroxytryptamine 7 Receptor Isoforms
Journal of Pharmacology and Experimental Therapeutics, 2005
The human 5-hydroxytryptamine 7 (5-HT 7) serotonin receptor is a class A G-protein coupled receptor that has three isoforms, 5-HT 7(a) , 5-HT 7(b) , and 5-HT 7(d) , which are produced by alternative splicing. The 5-HT 7 receptors are expressed in discrete areas of the brain and in both vascular and gastrointestinal smooth muscle. Central nervous system 5-HT 7 receptors may play a role in mood and sleep disorders. 5-HT 7 receptors show high affinity for a number of antidepressants and typical and atypical antipsychotics. We report here that the human 5-HT 7(d) isoform expressed in human embryonic kidney (HEK) 293 cells exhibits a pattern of receptor trafficking in response to agonist that differ from 5-HT 7(a) or 5-HT 7(b) isoforms. We employed a modification of a live cell-labeling technique to demonstrate that surface 5-HT 7(d) receptors are constitutively internalized in the absence of agonist. This is in contrast to 5-HT 7(a) and 5-HT 7(b) isoforms, which do not show this profound agonistindependent internalization. Indeed, the 5-HT 7(d) isoform displays this internalization in the presence of a 5-HT 7-specific antagonist. In addition, the human 5-HT 7(d) isoform shows a diminished efficacy in stimulation of cAMP-responsive reporter gene activity in transfected cells compared with 5-HT 7(a) or 5-HT 7(b) receptors expressed at comparable levels. Thus, the carboxy-terminal tail of 5-HT 7(d) , which is the longest among known human 5-HT 7 isoforms, may contain a motif that interacts with cellular transport mechanisms that is distinct from 5-HT 7(a) and 5-HT 7(b). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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.
The 5-HT1A receptor: an overview of recent advances
Neurochemical Research, 1991
Progress in the field of neuronal receptor research has accelerated during the last few years due to developments in pharmacology and molecular biology. This is particularly true in the case of the serotonin 5-HTIA receptor. In 1983 the very selective, high affinity 5-HTla agonist 8-OH-DPAT was developed which allowed the pharmacology and distribution of the 5-HT1A receptor in the central nervous system of the rat and man to be extensively characterized. By 1987, the gene encoding this receptor protein was cloned and sequenced, allowing not only elucidation of its structure, but also better insight into the nature of its coupling to transmembrane signal transduction systems. Thus in a short period of time considerable knowledge has accumulated on how serotonin exerts its functions in the central nervous system via the 5-HT1A receptor. In the present review we will briefly discuss some of the latest developments regarding the 5-HT:A receptor.