The 5-HTR4 gene is 172,607 bp long (according to UCSC, GRCh38/hg38), located on the minus (-) strand and spans 12 exons (NCBI Homo sapiens Annotation Release 109).

The gene has 13 transcripts (Table 1)
Table 1. Transcripts of the human 5-HTR4 gene (Ensemble, GRCh38.p12).

Name	Transcript ID	bp	Protein (aa)	Biotype
HTR4-201	ENST00000360693.7	3082	428	Protein coding
HTR4-202	ENST00000362016.6	2979	428	Protein coding
HTR4-203	ENST00000377888.7	2962	388	Protein coding
HTR4-209	ENST00000521530.5	1323	387	Protein coding
HTR4-210	ENST00000521735.5	1242	378	Protein coding
HTR4-207	ENST00000520514.5	1236	411	Protein coding
HTR4-213	ENST00000631296.1	1224	388	Protein coding
HTR4-204	ENST00000517929.5	1201	360	Protein coding
HTR4-206	ENST00000520086.1	2272	87	Protein coding
HTR4-211	ENST00000522588.5	1326	378	Nonsense mediated decay
HTR4-212	ENST00000524063.3	2274	371	Nonsense mediated decay
HTR4-205	ENST00000519495.1	869	No protein	Processed transcript
HTR4-208	ENST00000521124.5	1221	No protein	Retained intron

The product of HTR4 gene (5q32) is 5-HT4 receptor (Ohtsuki et al., 2002), weight as 43.761 Dalton with a length of 388 amino acids (Blondel et al., 1997; Claeysen et al., 1997; Van den Wyngaert et al., 1997). The protein has 9 known isoforms produced by alternative splicing, but the potential isoforms seem to exist (https://www.uniprot.org/uniprot/Q13639). Isoform 5-HT4(B) (Q13639-1) was considered as canonical sequence and provided in Figure 1. The translated glycoprotein is a member of the serotonin receptor family which are induced in response to the presence of serotonin via G-protein coupling process. In general, the 5-HT4 receptor is a glycosylated transmembrane protein expressed in both CNS and peripheric nervous system (PNS) that acts as a modulator in the release of various neurotransmitters (NCBI). It expressed in various tissues/cells such as the brain, esophagus, ileum, colon, bladder, heart, and adrenocortical cells. In the brain, they are particularly expressed in the hypothalamus, nucleus accumbens, amygdala, Calleja islands, olfactory tubercle, fundus striatum, ventral pallidum, septum, hippocampus, and basal ganglia including substantia nigra (Bockaert et al., 2008).

5-HTR4 is expressed in different human tissues/organs but mostly in gastrointestinal tract, brain, and muscles in descending order. Please see The Human Protein Atlas (http://www.proteinatlas.org)

5-hydroxytryptamine (5-HT or serotonin) is a crucial neurotransmitter of CNS with highly conserved monoamine structure (Hodge et al., 2013). It plays a role in different physiological events of CNS and PNS by interacting with more than one receptor subtype (Cichon et al., 1998). These countless functions are actualized by seven 5-HT subtype receptors (Kroeze et al., 2002; Hodge et al., 2013). The 5-HTR4 is a G-protein-coupled receptor that arouses adenylate cyclase as a primary mode of signal transduction. By the arousal of adenylate cyclase, the concentration of cAMP elevates (Lalut vd., 2017). However, 5-HTR4 refers to both G protein-dependent and G protein-independent pathways (Figure 2a-b). The main G protein introduced by 5-HTR4 signaling is the Gs (shown in Figure 2a) that leads to the activation of the cAMP/ PKA pathway (Barthet et al., 2005). The G-protein independent non-canonical pathway activates Src (shown in Figure 2b) and subsequently ERK kinases, causing pERK1/ 2 phosphorylation (Barthet et al., 2007).
This receptor was first described in primary cultures of mouse embryonic colliculus neurons based on 5-HT-induced cAMP production (Dumuis et al., 1988). It plays potential roles in the physiology of cardiovascular, urinary, and endocrine systems. It is also implicated in the pathophysiology of diseases occurring in adrenal glands as well as urinary and gastrointestinal systems (Ford and Clarke., 1994). 5-HTR4s are responsive in the events occurring in adrenal glands, colon, cortex (Monferini et al.,1993), and atrial appendages (Kaumann et al., 1990, 1991; Turconi et al., 1991; Quadid et al., 1992) of human tissues by encouraging cAMPs. In turn, cAMP inductions lead to steroid secretion in adrenal glands (Lefebvre et al., 1992) and circular muscle contraction in the colon (Tam et al.,1992).
Therapeutic agents interacting with 5-HTR4
Advances in research on 5-HT set forward the development of therapeutics selectively interacting with 5-HT receptors. In the present day, these ligands mainly divided into 5 categories/classes. They are indoles including 5-HT analogs, indole carboxylates, and indole carboxamides, benzamides, benzoates, aryl ketones, and benzimidazoles (Bockaert et al., 2004).
First-class 5-HTR4 agonists include substituted tryptamines or 5-HT agonists next to the substituted triple-carbazimidamides. 5-MeOtryptamine is an interesting vehicle due to the lack of its affinity to 5-HTR3 (Craig et al., 1990). The indole carbazimidamide derivative, HTF 919 (Tegaserod), has been found to be a potent partial agonist with high affinity to 5-HTR4. In vivo studies show that Tegaserod enhances gastrointestinal motor activity and adjusts impaired motility throughout the gastrointestinal tract. (Bockaert et al., 2004). Tegaserod is a benzamide derivative which was approved by the FDA and other regulatory agencies for the treatment of women with inflammatory bowel disease (IBD) in which the constipation is predominant or functional constipation (Tonini and Pace 2006; Shin et al., 2015). However, it was later withdrawn from the market due to possible cardiovascular side-effects (Chey et al., 2008; Thompson 2007). Three novel 5-HTR4 agonists (Prucaloprid, Naronapride, and Velusetrag) have therapeutic potential for patients with IBD. These alternative drugs have a higher affinity for 5-HTR4 compared to Tegaserod (Halland and Talley, 2013). YKP10811 is a mucosal partial agonist for the 5-HTR4, induces serotonin release, initiates peristaltic reflex, and has a low cardiovascular adverse effect. Unlike traditional prokinetic drugs such as Tegaserod, YKP10811 exhibits an antinociceptive effect on inflammation as well as acute stress-associated colonic hypersensitivity, and also considered as a candidate drug for IBD (Gilet et al., 2014).
The second class of 5-HTR4 agonists includes first-generation benzamides carrying 2-methoxy-4-amino-5-chloro substitution (Dumuis et al., 1989). These benzamides (zacopride, renzapride, and metoclopramide) were found to be non-selective and always cause antagonistic activity at the 5-HTR3 (Bockaert et al., 2004).
The third class of agonists, so-called benzoate derivatives were deliberated with the replacement of benzamides amide bond with an ester one, resulting in increased affinity for 5-HTR4. The first partial and selective drug of this series is ML 10302, which has a high affinity for 5-HTR4 and poor affinity for other 5-HT receptor subtypes including 5-HTR3 (Langlois et al., 1994; Bockaert et al., 2004). Moser and colleagues (2002) synthesized SL65.0155, a novel compound with high affinity for 5-HTR4. Being a benzodioxane derivative, it is particularly effective for learning and memory (Moser et al., 2002).
The fourth class is consist of benzimidazoles (BIMU 8 and BIMU 1), which are potent and effective 5-HTR4 agonists entering CNS (Dumuis et al., 1991; Rizzi et al., 1992). To overcome their metabolic variability, aryl ketones were prepared with 5-HTR4 ester ligands; instead of the ester linkage of the antagonist RS 23597, a partial agonist RS 17017 was synthesized, which has a similar affinity for 5-HTR4 (Clark et al., 1994). Increasing the size of the alkyl group led to an increase in the activity of the agonists RS 67333 and RS 67506 (Eglen et al., 1995). RS 67333, a selective 5-HTR4 partial agonist, is known to easily cross the blood-brain barrier. Systemic administration of selective 5-HT4 receptor partial agonists, like RS 67333 and RS 17017, enhances rodent performance in olfactory relational learning (Marchetti et al., 2004), social-spatial memory tests (Letty et al., 1997), and also improves delayed sample matching in young/ old macaques (Terry et al., 1998). RS 67333 has been shown to inhibit β-amyloid peptide formation in primary cortical neurons (Cho and Hu, 2007). Interestingly, RS 67333 produces a rapid antidepressant effect after only three days of administration to rodents (Lucas et al., 2007). A more recent study comparing RS67333 with antidepressant drug Fluoxetine (FLX) confirmed that RS67333 causes anxiolytic-like effects after only 7 days in several behavioral tests and that 5-HTR4 agonists produce faster effects than currently used antidepressants (Mendez-David et al., 2014). Another interesting agonist is a pyridine carboxamide, a cognitive drug (VRX-03011) for Alzheimers disease (Bockaert et al., 2008).
Fifth class includes indoles such as GR 113808 with high affinity for 5-HTR4, and low affinity for 5-HTR3 (Gale et al., 1994). Further, [3H] GR 113808 is the first commercially available radioligand for 5-HTR4 affinity studies (Grossman et al., 1993). SB 203186 is an indole ester with potent and selective 5-HTR4 antagonistic properties in various physiological assays, but the short half-life limits its in vivo application (Parker et al., 1993). In contrast, SB 207266 is a highly potent and selective antagonist with a long duration of action after oral administration (Gaster et al., 1995). SB 207266 is a very useful vehicle in CNS studies (Wardle et al., 1996; Gaster and King 1997).
Three selective and high-affinity 5-HTR4 antagonists (GR 125487, SB 207266, and ML 10375) have been reported to exhibit reverse agonistic activity (Claeysen et al., 2000; Blondel et al., 1998). Roche Bioscience generated three potent 5-HTR4 reverse agonists, RO 116-2617, RO 116-0086, and RO 116-1148 (Joubert et al., 2002). 5-HTR4 ligands that may be of interest in CNS studies are listed in Table 2.
Table 2. 5-HTR4 ligands used in CNS studies (Adopted from Bockaert et al., 2004).

Agonists	Antagonists	Reverse Antagonists
5-HT	GR 113308	RO 116-2617
5-MeOT	GF 125487	RO 116-0086
HTF919	SB 203186	RO 116-1148
Cisapride	LY 353433
SC 53 116	SB 207 266
SB 205 149	SB 204070
Prucalopride	RS 23597
Mosapride	ML 10375
Y-34959	ATBI 221
ML10302	RS 39606
LS-65 0155	RS 67532
RS 67333	RS 100235
RS 67506	RS 100302
BUMI 1
BUMI 8

Table 3. Pairwise alignment of 5-HTR4 gene protein sequences (in distance from human) (HomoloGene, NCBI).

Pairwise Aligment Scores
Gene		Identity (%)
Species	Symbol	Protein	DNA
H.sapiens	HTR4
vs. P.trogladytes	HTR4	99,8	99,6
vs. M.mulatta	HTR4	98,6	98,1
vs. C.lupus	HTR4	96,6	93,6
vs. B.taurus	HTR4	94,8	93,7
vs. M.musculus	Htr4	93,3	90,9
vs. R. norvegicus	Htr4	92,7	90,6
vs. G. gallus	HTR4	88,5	82,6
vs. X. tropicalis	LOC100493952	83,4	74,5
vs. D. rerio	LOC556843	75,3	71,9
vs. D. melanogaster	Oa2	47,5	54,3

A list of 5-HTR4 mutations in cancer can be found in COSMIC, the Catalogue Of Somatic Mutations In Cancer, https://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=HTR4. According to the Human Protein Atlas, HTR4 has been found strongly expressed in prostate and endometrial cancers.