Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-09-19
2003-01-14
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C435S007100, C435S007200, C435S007210
Reexamination Certificate
active
06506580
ABSTRACT:
The present invention relates to novel splicing variants of the serotoninergic receptor 5-HT
4
in humans.
BACKGROUND OF THE INVENTION
Serotonin (5-hydroxytryptamine or 5-HT) is a neurotransmitter which is located in the central and peripheral nervous system of vertebrates, where it exerts various physiological roles mediated by different receptor subtypes (Saxena, 1995). The receptors 5-HT
4
represent a member of the family of receptors with seven transmembrane (7TM) domains coupled to a G protein which is positively coupled to adenylate cyclase (Hedge and Eglen, 1996). The receptors 5-HT
4
are expressed in a wide variety of tissues, including the human brain and the rodent brain (Eglen et al., 1995), the human, dog, pig and rodent gastro-intestinal tract, and the pig and human heart (Hedge and Eglen, 1996). In the mammalian brain, the receptors 5-HT
4
contribute to dopamine secretion (Bonhomme et al., 1995) and regulate learning and long-term memory via the modification of acetylcholine release (Marchetti-Gauthier et al., 1997). In the peripheral tissues, the receptors 5-HT
4
have proven to regulate gastro-intestinal tract motility, intestinal electrolyte secretion, adrenal secretion of corticosteroides, bladder contraction and atrium contractility (Edge and Eglen, 1996).
The receptors 5-HT
4
are involved in a wide variety of central and peripheral disorders, including cardiac arrhythmias (Kaumann, 1994) and neurodegenerative disorders (Reynolds et al., 1996; Wong et al., 1996). In addition, the development of receptor 5-HT
4
agonists and antagonists may have therapeutic applications in the central nervous system for treating neuropsychiatric disorders associated with a dysfunction of the central dopaminergic system, such as Parkinson's disease (Bonhomme et al., 1995), or for treating amnesic deficiencies as presented in patients suffering from Alzheimer's disease (Marchetti-Gauthier et al., 1997). Such medicines might also be useful for treating peripheral disorders such as irritable bowel syndrome, gastroparesia, urinary incontinence and cardiac arrhythmias (Hedge and Eglen, 1996).
The receptors 5-HT
4
present a unique pharmacology which is clearly different from that of the other members of the 5-HT receptor family (Ford and Clarke, 1993). Most pharmacological and transductional studies on the receptors 5-HT
4
have been carried out on the central nervous system and gastro-intestinal tract of rodents and in porcine and human hearts (Eglen et al., 1995; Hedge and Eglen, 1996). Although the pharmacology of the receptors 5-HT
4
present in these preparations is very similar, unexplained differences exist. Thus, benzamides, such as renzapride and cisapride, behave as potent and total agonists of the receptors 5-HT
4
in mouse colliculi neurons, but are less potent and only partial agonists in the human heart (Ford and Clarke, 1993; Hoyer et al., 1994) and the detrusor muscle isolated from human bladder (Ford and Clarke, 1993, Hoyer et al., 1994; Candura et al., 1996). 5-Methoxytryptamine (5-MeOT), which is a 5-HT
4
agonist, has an unusually weak agonist action on the receptors 5-HT
4
of the human detrusor muscle (Candura et al., 1996). ML10302, an agonist of the receptor 5-HT
4
which imitates the effect of 5-HT on the relaxation of rat oesophagus, and on electrically-induced contraction in guinea pig ileum (Langlois et al., 1994), has a weak agonist effect combined with a net antagonism of 5-HT-on the cAMP response generated by the human receptor 5-HT
4(a)
cloned from human atrium (Blondel et al., 1997), and an antagonist effect in colliculus neurons (Ansanay et al., 1996). In addition, the desensitization mechanisms for the receptors 5-HT
4
are tissue-dependent. Specifically, a homologous rapid and total desensitization (cAMP-independent) of the receptors 5-HT
4
is observed in mouse colliculi neurons (Ansanay et al., 1996) and rat oesophagus (Ronde et al., 1996), whereas this type of receptor is desensitized to a lesser degree in the human atrium (Kaumann et al., 1991).
The first receptor 5-HT
4
was cloned from rat brain (Gerald et al., 1995), and two splicing variants (r5-HT
4S
and r5-HT
4L
) were identified. These variants differ in the lengths and the sequence of the carboxy-terminal end. The long form (r5-HT
4L
), which has also been cloned in mouse colliculi neurons (m5-HT
4L
), has transcripts in approximately each part of the brain (Claeysen et al., 1996). An interesting observation comes from the peripheral distribution of the transcripts of r5-HT
4L
and r5-HT
4s
in rats. Whereas both forms are expressed in the gastro-intestinal tract (ileum and colon), only the r5-HT
4s
transcript is found in the heart (Gerald et al., 1995). In addition, Sr-HT
4s
has been found exclusively in the atrium (Gerald et al., 1995). The human homologues of the receptors r5-HT
4s
and r5-HT
4L
, herein termed h5-HT
4(a)
and h5-HT
4(b)
, have recently been cloned. The receptor h5-HT
4(a)
was cloned from human heart (Blondel et al., 1997; Claeysen et al., 1997); the receptor h5-HT
4(b)
was cloned from a library (Van de Wyngaert et al., 1997). These two receptors h5-HT
4(a)
and h5-HT
4(b)
, when transiently expressed in COS-7 cells, present a conventional 5-HT
4
pharmacological profile. However, the affinities of the cloned receptor h5-HT
4(a)
for agonists such as ML10302, BIMU1, renzapride and zacopride are lower than those found in the brain.
The inventors have now shown the existence of two novel serotonin receptor subtypes in humans, termed receptors h5-HT
4(c)
and h5-HT
4(d)
.
The analysis of the sequence homologies suggests that forms h5-HT
4(c)
and h5-HT
4(b)
are respectively the human correspondents of forms r5-HT
4s
and r5-HT
4L
in rats, whereas forms h5-HT
4(c)
and h5-HT
4(d)
represent two novel isoforms of the receptor. The isoform h5-HT
4(c)
presents a high number of putative phosphorylation sites (one for protein kinase C, one for protein kinase A/protein kinase G and two for casein kinase II), all contained in the last 25 residues of the amino acid sequence.
The phosphorylation of the carboxy-terminal end of seven-transmembrane-domained receptors by protein kinase A regulates the desensitization of the receptor and the association of the receptor with G protein in response to increasing concentrations of cAMP (Dohlman et al., 1991).
In addition, in seven-transmembrane-domained receptors such as the &bgr;
1
- and &bgr;
2
-adrenergic receptors (Pei et al., 1994; Freedman et al., 1995) and the receptor 5-HT
2c
(Westphal et al., 1995), the phosphorylation of the receptor by non-cAMP-dependent protein kinases such as the &bgr;-adrenergic receptor kinases (&bgr;ARK1 and &bgr;ARK2) or rhodopsin kinase allows substrate-activated homologous desensitization. The number and nature of the phosphorylation sites present at the C-terminal end of the receptor 5-HT
4
splicing variants are thus capable of influencing the negative regulation of receptor function.
Tissue-dependent differences in receptor 5-HT
4
desensitization mechanisms have been reported (Ford and Clarke, 1993), and may be related to the restricted profiles of the expression of the receptor 5-HT
4
isoforms in the various tissues. Thus, the receptor 5-HT
4
desensitization mechanism in mouse colliculi neurons resembles that described for &bgr;-adrenergic receptors, and appears to be independent of the cAMP pathway. It has been proposed that a “&bgr;ARK-like” kinase mediates the specific phosphorylation of the receptor 5-HT
4
in these neuronal cells (Ford and Clarke, 1993). C-terminal phosphorylation of the isoform 5-HT
4(c)
by casein kinase II or PKC might thus explain the cAMP-independent desensitization observed in these cells.
The form 5-HT
4(d)
is characterized by a very short C-terminal end, with a coding sequence which finishes two amino acids after Leu
358
. Similarities in structure between the 5-HT
4
C-terminal ends and the 5-HT
7
C-terminal end (same number of variants, including a phosphorylation site-rich isoform and a truncated isoform close to the splicing site), de
Blondel Olivier
Dahmoune Yamina
Fischmeister Rodolphe
Gastineau Monique
Hoebeke Johan
Institut National de la Sante et de la Recherche Medicale (INSER
Larson & Taylor PLC
Ulm John
LandOfFree
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