Elsevier

Neuropharmacology

Volume 39, Issue 8, July 2000, Pages 1451-1462
Neuropharmacology

Cloning, expression and pharmacological characterisation of the mouse somatostatin sst5 receptor

https://doi.org/10.1016/S0028-3908(00)00063-0Get rights and content

Abstract

The mouse somatostatin (somatotropin release inhibiting factor, SRIF) sst5 receptor coding sequence was cloned from a mouse BALB/c genomic library. It shows 97% and 81% homology with the corresponding rat and human receptors, respectively. The msst5 receptor messenger RNA (mRNA) is present at low levels in the adult mouse brain, with significant expression in a few nuclei only, e.g. in the septum (lateral septal nuclei) or the amygdala (medial amygdaloid nucleus); very few signals were observed in the mesencephalon, metencephalon, and myelencephalon (except the dorsal motor nucleus of the vagus nerve).

The msst5 receptor was stably expressed in the hamster fibroblast cell line CCL39-SRE-Luci, which harbours the luciferase reporter gene driven by the serum responsive element. [125I]LTT-SRIF-28 ([Leu8, D-Trp22, 125I-Tyr25]-SRIF-28), [125I]Tyr10-CST, [125I]CGP 23996, and [125I]Tyr3-octreotide labelled msst5 receptors with high affinity (pKd values: 11.0, 10.15, 9.75 and 9.43) and in a saturable manner, but defined different Bmax values: 697, 495, 540 and 144 fmoles/mg, respectively. [125I]LTT-SRIF-28-labelled sites displayed the following rank order: SRIF-28> rCST-14> somatuline > CGP-23996= SRIF-14= octreotide, whereas [125I]Tyr3-octreotide-labelled sites displayed a different profile: octreotide > SRIF-28> rCST-14= somatuline > SRIF-14> CGP-23996. The pharmacological profiles determined with [125I]LTT-SRIF-28, [125I]CGP 23996 and [125I]Tyr10-CST correlated highly significantly (r2 =0.88–0.99), whereas [125I]Tyr3-octreotide binding was rather divergent (r2 =0.77). Also, human and mouse sst5 receptor profiles are very different, e.g. r2 =0.385 for [125I]Tyr10-CST and r2 =0.323 for [125I]LTT-SRIF-28-labelled sites.

Somatostatin induces expression of luciferase reporter gene in CCL39-SRE-Luci cells. The profile was consistent with a msst5 receptor-mediated effect although apparent potency in the luciferase assay was much reduced compared to radioligand binding data: Octreotide = SRIF-28> rCST-14= SRIF-14= CGP-23996. Octreotide, SRIF-28, BIM23052 and D Tyr Cyanamid 154806 behaved as full or nearly full agonists in comparison to SRIF-14, whereas the other compounds had relative efficacies of 40 to 70%.

The present study shows that agonists radioligands define apparently different receptor populations in terms of number of sites and pharmacological profile in cells expressing a single recombinant receptor. These variations suggest that the conformation of the ligand receptor complex may vary depending on the agonist. Further, the msst5 receptor, although primarily coupled to Gi/Go proteins, is able to stimulate luciferase gene expression driven by the serum responsive element. Finally, it is suggested that putative sst2 selective agonists e.g. octreotide, RC160 or BIM23027 show similar or higher potency at msst5 receptors than SRIF-14.

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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    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.

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