Cloning, expression and pharmacological characterisation of the mouse somatostatin sst(5) receptor.
The mouse somatostatin (somatotropin release inhibiting factor, SRIF) sst(5) 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 msst(5) 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 msst(5) 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. [(125)I]LTT-SRIF-28 ([Leu(8), D-Trp(22), (125)I-Tyr(25)]-SRIF-28), [(125)I]Tyr(10)-CST, [(125)I]CGP 23996, and [(125)I]Tyr(3)-octreotide labelled msst(5) receptors with high affinity (pK(d) 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. [(125)I]LTT-SRIF-28-labelled sites displayed the following rank order: SRIF-28> rCST-14> somatuline > CGP-23996= SRIF-14= octreotide, whereas [(125)I]Tyr(3)-octreotide-labelled sites displayed a different profile: octreotide > SRIF-28> rCST-14= somatuline > SRIF-14> CGP-23996. The pharmacological profiles determined with [(125)I]LTT-SRIF-28, [(125)I]CGP 23996 and [(125)I]Tyr(10)-CST correlated highly significantly (r(2) =0.88-0.99), whereas [(125)I]Tyr(3)-octreotide binding was rather divergent (r(2) =0.77). Also, human and mouse sst(5) receptor profiles are very different, e. g. r(2) =0.385 for [(125)I]Tyr(10)-CST and r(2) =0.323 for [(125)I]LTT-SRIF-28-labelled sites. Somatostatin induces expression of luciferase reporter gene in CCL39-SRE-Luci cells. The profile was consistent with a msst(5) 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 msst(5) 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 sst(2) selective agonists e.g. octreotide, RC160 or BIM23027 show similar or higher potency at msst(5) receptors than SRIF-14.
Feuerbach
D
D
Nervous System Research, S-386/745, Novartis Pharma AG, CH-4002, Basel, Switzerland.
Fehlmann
D
D
Nunn
C
C
Siehler
S
S
Langenegger
D
D
Bouhelal
R
R
Seuwen
K
K
Hoyer
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D006801:Humans; D017403:In Situ Hybridization; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008297:Male; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D015282:Octreotide / Q000378:metabolism / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011869:Radioligand Assay; D017481:Receptors, Somatostatin / Q000819:agonists / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013004:Somatostatin / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
C091737:BIM 23056; D009842:Oligopeptides; D010456:Peptides, Cyclic; D012333:RNA, Messenger; D017481:Receptors, Somatostatin; D011993:Recombinant Fusion Proteins; D013004:Somatostatin; C040968:seglitide; C094573:somatostatin receptor 5; D004247:DNA; D008156:Luciferases; D015282:Octreotide