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Molecular Cloning of a Somatostatin-28 Receptor Proc. Nail. Acad. Sci. USA Vol. 89, pp. 10267-10271, November 1992 Biochemistry Molecular cloning of a somatostatin-28 receptor and comparison of its expression pattern with that of a somatostatin-14 receptor in rat brain (neuropeptide receptor/in situ hybridization/neuron/astrcyte/release-inhibiting factor) WOLFGANG MEYERHOF, IRIS WULFSEN, CHRISTIANE SCHONROCK, SUSANNE FEHR, AND DIETMAR RICHTER Institut fur Zelibiochemie und klinische Neurobiologie, Universitits-Krankenhaus Eppendorf, Universitit Hamburg, Martinistrasse 52, 2000 Hamburg 20, Federal Republic of Germany Communicated by Floyd E. Bloom, June 16, 1992 (receivedfor review March 27, 1992) ABSTRACT The tetradecapeptide somatotropin-release fashion and can cause either depolarization (9) or hyperpo- inhibiting factor somatostatin-14 regulates the release of pep- larization (10). Less is known about the functional role ofthe tide hormones and also functions as neurotransmitter. The octacosapeptide that shows potencies different from those of octacosapeptide somatostatin-28, the N-terminally extended the tetradecapeptide in controlling hormone secretion from form of somatostatin-14, shows similar biological activities yet target tissues. Also, somatostatin-28 appears to bind to with different potencies. Both peptides most likely function receptors distinct from those that bind somatostatin-14, sug- through distinct receptors. Here we report on the molecular gesting at least two subtypes of somatostatin receptors (11). and functional characterization of a somatostatin-28 receptor Raynor et al. (12) characterized two receptor subtypes that (SSR-28) cloned from a rat brain cDNA library. The nucleotide mediate their activities via GTP-binding proteins, by inhib- sequence contains an open reading frame for a protein of 428 iting adenylate cyclase and by modulating Ca2+ currents; one amino acid residues with a predicted molecular mass of47 kDa. subtype additionally potentiates a delayed rectifier potassium Binding assays using radiolabeled somatostatin-14 and mem- current. cDNAs encoding two structurally different periph- branes from COS cells transfected with the cloned cDNA show eral somatostatin receptors, somatostatin receptors 1 and 2 that this receptor, SSR-28, has a higher binding affinity for (SSTR1 and SSTR2, respectively), have recently been cloned somatostatin-28 (IC5. = 0.24 nM) than for somatostatin-14 from mice and humans (1). They preferentially bind somato- (ICso = 0.89 nM). RNA blot analysis reveals a 4.4-kilobase statin-14, having less affinity for somatostatin-28. The de- mRNA in rat cerebellum and at significantly lower abundance duced amino acid sequence of SSTR1 is nearly identical to in other brain regions. In situ hybridization indicates that the previously identified rat orphan receptor rGHJP se- SSR-28 mRNA is present in the granular and Purkiqje cell quence (97% identity; ref. 13), herein we will refer to this layers ofthe cerebellum and in the large cells ofthe hypoglossal receptor as somatostatin-14 receptor (SSR-14). Here we nuclens of the brain stem. Signals for SSR-28 mRNA do not report the molecular cloning of a cDNA encoding a somato- overlap with those of a previously cloned rat receptor that statin receptor, termed somatostatin-28 receptor (SSR-28), preferentially binds somatostatin-14 (SSR-14). SSR-14 mRNA that preferentially binds the octacosapeptide.* The corre- is found in the medial cerebellar nucleus, horizontal limb ofthe sponding mRNA is found in distinct neural cell populations diagonal band, various hypothalamic nuclei, and in layers IV that are different from those containing the rat tetradecapep- and V of the cortex. In the rat cerebellum, SSR-14 and SSR-28 tide receptor. mRNAs are developmentally regulated; the levels ofthe former are highest around birth and levels of the latter are highest at MATERIALS AND METHODS the adult stage. Materials. Ifnot otherwise stated 3-month-old male Wistar The somatostatin peptide hormone family is composed of at rats were used. Peptides and analogs were obtained from least two functionally active peptides, a tetradecapeptide Bissendorf (Hannover, F.R.G.) or Bachem. RNA blot anal- (somatostatin-14) and an N-terminally elongated form that ysis (13, 14) and in situ hybridization (15) were carried out as consists of 28 amino acid residues (somatostatin-28). Both reported. peptide hormones are derived from a common prohormone Cloning of a cDNA Encoding SSR-28. Preparation of total precursor through tissue-specific proteolytic cleavage (2). cellular RNA, isolation of poly(A)+ RNA, and cDNA syn- Somatostatin-14 was initially isolated from ovine hypothal- thesis were carried out according to standard protocols (14). ami on the basis of its ability to inhibit the release of growth Receptor-encoding cDNA frgments were amplified using hormone from the anterior pituitary (3). It was subsequently primers and conditions as reported (16) and, after subcloning found to inhibit the release of other peptide hormones from into M13mpl8 vectors, were submitted to nucleotide se- the pituitary (prolactin and thyrotropin) (4) and from the quence analysis (17). A rat brain cDNA library in A ZAP pancreas (glucagon and insulin) (5, 6), to control the secretion (Stratagene) was plated at a density of5 x 104 plaque-forming of gut hormones and gastrointestinal motor activity, and to units per 15-cm diameter plate and phage DNA was trans- decrease nutrient absorption from the gut (7). Within the ferred to nylon membranes (Amersham). The filters were central nervous system, somatostatin-14 is assumed to be a hybridized with the amplified cDNA fragments labeled in the neurotransmitter or modulator, based on depolarization ex- presence of [a-32P]dCTP (specific activity, 3000 Ci/mmol; 1 periments of somatostatinergic nerve cells that cause peptide Ci = 37 GBq; Amersham; ref. 18). Membranes were finally release in a Ca2+-dependent fashion (8). At the cellular level, washed for 30 min in 0.2x standard saline citrate at 650C. somatostatin-14 influences neuronal firing rates in a complex Abbreviations: SSR-14 and SSR-28, receptors for somatostatin-14 and -28, respectively; SSTR1 and 2, somatostatin receptors 1 and 2, The publication costs of this article were defrayed in part by page charge respectively, as described in ref. 1. payment. This article must therefore be hereby marked "advertisement" *The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. X63574). 10267 Downloaded by guest on October 3, 2021 10268 Biochemistry: Meyerhof et al. Proc. Natl. Acad Sci. USA 89 (1992) Positive phages were plaque-purified and the receptor- still lacks roughly 400 nucleotides as judged from the size of encoding plasmids were excision-rescued from A ZAP clones the corresponding 4.4-kb mRNA. by superinfection with R-408 helper phages (14). Fragments The deduced amino acid sequence displays features typical of the cDNAs, generated by restriction digests or DNase I of guanine nucleotide binding-protein-coupled receptors, deletions (19), were sequenced as reported (13). such as seven hydrophobic segments of 20-27 amino acid Ligand Binding Assays. The 3.5-kilobase-pair EcoRP frag- residues (Fig. 1). Furthermore, there are two potential ment encoding SSR-28 was subcloned into the corresponding N-linked glycosylation sites, Asn-18 and Asn-31, in the site of pcDNAI (Invitrogen); this construct was used to trans- N-terminal extracellular portion of the protein and two con- fect COS-7 cells by the calcium phosphate/glycerol method served cysteine residues (positions 117 and 192) in the first (14). Membrane fractions were prepared from 2.4 x 107 cells, and second extracellular loop (22). The deduced amino acid homogenized in 20 ml of buffer A (50 mM Tris-HCl, pH 7.6/1 sequence also contains several consensus sites for phosphor- mM EDTA/1 mM dithiothreitol/0.1 mM phenylmethylsulfo- ylation (23) by cAMP-dependent protein kinase (Ser-260), nyl fluoride), centrifuged, and taken up in 800 /ul of buffer A protein kinase C (Ser-260), or multifunctional calmodulin- (20). For ligand binding assays, 40 ,ug ofmembrane protein (1.5 dependent protein kinase II (Ser-75, Ser-245, Ser-260, and mg/ml) was incubated with 50 pM [[125I]Tyr1]somatostatin-14 Ser-346). (2200 Ci/mmol; NEN) in 300 ,ul of binding buffer [50 mM Functional Identification. Alignment of the amino acid Hepes, pH 7.5/5mM MgCl2/Trasylol (200 kallikrein inhibition sequence deduced from the rat cDNA clone with other units/mil; Bayer, Leverkusen, F.R.G.)/bacitracin (0.02 ,ug/ receptor protein sequences revealed -50%o identity with ml; Sigma)/bovine serum albumin (10 mg/ml)/phenylmethyl- SSTR1 and SSTR2, the cloned somatostatin receptors from sulfonyl fluoride (0.02 ,ug/ml)] for 1 hr at room temperature in mice and humans (1) and the rat orphan receptor rGHJP (13) the presence or absence ofunlabeled somatostatin-14 or -28 at (Fig. 1) that we refer to as SSR-14. To demonstrate that the concentrations as indicated. cloned cDNA indeed encodes a putative somatostatin recep- tor, ligand-binding assays were performed using membranes ofCOS-7 cells that transiently express the clone. Fig. 2 shows RESULTS AND DISCUSSION the binding of iodinated somatostatin-14. Binding of radioli- cDNA Cloning and Structure of a Rat Somatostatin Recep- gand was competed by using unlabeled somatostatin-14 and tor. cDNA fragments were generated by PCR amplification -28 with inhibitory concentrations for half-maximal response
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