Cloning, expression and pharmacological characterisation of the mouse somatostatin sst5 receptor
Introduction
The cyclic peptide somatostatin (SRIF, somatotropin release inhibiting factor) exists in two different forms, namely with 14 amino acids (SRIF-14) and in an N-terminally extended form with 28 amino acids (SRIF-28). Five different receptors for SRIF (sst1 to sst5, Hoyer et al., 1994, Hoyer et al., 1995, Bell and Reisine, 1993) which all belong to the G-protein coupled receptor family have been cloned from various species including rat and man. They are grouped in two classes according to structure and pharmacology (Hoyer et al., 1995): sst2, sst3 and sst5 with high affinity for octreotide, seglitide and somatuline and sst1/sst4 with very low affinity for these short analogues of SRIF.
SRIF is a potent regulator of endocrine function by inhibiting the release of growth hormone from the pituitary, glucagon and insulin from the pancreas, and gastrin from the gut (Brazeau et al., 1972, Reichlin, 1983). SRIF has also neurological functions: it acts as a neurotransmitter and a neuromodulator in the central nervous system and peripheral tissues. In a number of neuropsychiatric disorders changes in brain somatostatin levels have been observed e.g. in epilepsy, meningitis, senile dementia, Alzheimer's disease and ACTH-dependent Cushing syndrome (Epelbaum, 1986).
Here we report the cloning of the mouse sst5 receptor, its pharmacological features in relation to the human receptor and its coupling via the SRE to luciferase in CCL39 cells.
Section snippets
PCR
Primers: the following primers were used for PCR, respectively sequencing analysis:
- msst5-241se
ATG GAG CCC CTC TCT TTG GC
- msst51340-as
TAC TGG GAC ACT CAA AGC CT
- se1
ATG TGG TGT TGC GGT ATG CC
- se2
GGT AGC CAA GCT GGC TAG TG
- se3
TGG TAG TGG TGC TGG TGT TC
- as1
AAG TAG AGG CCG GCA GAG GT
- as2
AAG ACC AAG AGC GGC AGA GA
- as3
GCC AGG TTC AGG ATG TAC AC
PCR using the PFU polymerase was carried out according to the manufacturer's protocol with 25 cycles and an annealing temperature of 60°C.
Screening of λ-phage library
The BALB/c genomic λ-fix
Cloning and expression of the mouse sst5 receptor
Cloning of the msst5 receptor fragment and transfection in CCL39 cells was carried out using standard procedures (see Methods). The comparison of our cloned sequence (msst5df) with the two reported sequences msst5 (Moldovan) and msst5hoch (Lublin et al., 1997) is shown in Fig. 1. The published sequences differ at 7 positions. At positions 141, 252, 267, 295 and 693 our sequence is identical to the sequence reported by Moldovan. At the position 909 where the Moldovan sequence displays an
Discussion
The present paper reports the cloning of the mouse sst5 receptor; the predicted peptide has 363 amino acids and shows 97 and 81% homology with the rat and human receptor. Differences with published sequences were observed, which may be attributed to strain (BALB/c compared to strain 129) and/or cloning.
The distribution of sst5 receptor mRNA studied in the mouse brain, confirms previous findings made in rat brain slices, i.e. that transcript levels are very low compared to those observed with
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Somatostatin 14
2007, xPharm: The Comprehensive Pharmacology ReferenceSomatostatin 28
2007, xPharm: The Comprehensive Pharmacology ReferenceSomatostatin receptor type 5 modulates somatostatin receptor type 2 regulation of adrenocorticotropin secretion
2005, Journal of Biological ChemistryCitation Excerpt :SST5, as previously reported for SST1 (31, 33), does not internalize, in agreement with previous reports suggesting either sub-membranal localization or enhanced trafficking to the membrane of SST5 after treatment with SRIF-14 (33, 38). An effect caused by the difference between human and mouse SST5 is possible; however, SRIF-28 was shown to bind mouse or human transfected SST5 with the same affinity (competing [125I]LTT-SRIF-28) (58, 59), human and mouse SST5 share 80% homology, and we also have demonstrated that hSST5 did not internalize in human cells. The results suggest functional interaction between SST2 and SST5 in AtT-20 cells.
Somatostatin inhibition of fictive respiration is modulated by pH
2004, Brain ResearchCitation Excerpt :In the adult mouse brain stem, mRNA of SSTR1 (SST receptor type 1) has been detected in the parabrachial nucleus, the locus coerulus, the NTS, the area postrema and the reticular formation, but no expression of the SSTR2 (SST receptor type 2) was detected [5]. The sst5 receptor mRNA is absent from the brain stem with the exception of the dorsal motor nucleus of the vagus nerve [17]. In the adult rat, sst2A receptor has been traced by immunohistochemistry in some nuclei of the pons and medulla [13,48] like the locus coerulus, parabrachial nucleus, NTS, lateral reticular nucleus and dorsal motor nucleus of the vagus, but no sst2B receptor was found [49].
The two subtype 1 somatostatin receptors of rainbow trout, Tsst <inf>1A</inf> and Tsst<inf>1B</inf>, possess both distinct and overlapping ligand binding and agonist-induced regulation features
2004, Comparative Biochemistry and Physiology - B Biochemistry and Molecular BiologyDeficiency of somatostatin (SST) receptor type 5 (SSTR5) is associated with sexually dimorphic changes in the expression of SST and SST receptors in brain and pancreas
2004, Molecular and Cellular Endocrinology
- 1
Present address: School of Biological Sciences, G 38 Stopford Building, Manchester University, Oxford Road, Manchester, M13 9PT, UK.
- 2
Present address: Department of Pharmacology, University of Pennsylvania, School of Medicine, 3620 Hamilton Walk (John Morgan Building), Philadelphia PA 19103, USA.