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Endocrinology Vol. 140, No. 12 5598-5608
Copyright © 1999 by The Endocrine Society

ARTICLES

Cloning of the Mouse Somatostatin Receptor Subtype 5 Gene: Promoter Structure and Function1

David F. Gordon, Whitney W. Woodmansee, Suzanne R. Lewis, R. Andrew James, William M. Wood and E. Chester Ridgway

Division of Endocrinology, Metabolism, and Diabetes (D.F.G., W.W.W., S.R.L., W.M.W., E.C.R.), University of Colorado Health Sciences Center, Denver, Colorado; and Department of Medicine (R.A.J.), University of Newcastle, Newcastle-upon-Tyne, United Kingdom

Address all correspondence and requests for reprints to: Dr. David F. Gordon, University of Colorado Health Sciences Center, Division of Endocrinology, Box B151, 4200 East Ninth Avenue, Denver, Colorado 80262. E-mail: david.gordon{at}uchsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Somatostatin is a peptide hormone whose actions are mediated by five somatostatin receptor subtypes (sst1–5). In the pituitary, somatostatin inhibits TSH release from thyrotropes and GH release from somatotropes. We have shown that sst5 transcripts and protein are induced by thyroid hormone in TtT-97 thyrotropic tumors. To map sequences responsible for promoter activity in pituitary cells, we cloned the mouse sst5 coding region of 362 amino acids and 12 kb of upstream DNA. Initial transfection studies in TtT-97 or GH3 cells mapped high levels of basal promoter activity to a 5.6-kb fragment upstream of the translational start, whereas shorter genomic fragments had low activity. To identify the transcriptional start site we used 5' RACE with TtT-97 poly A+ RNA and a sst5 antisense coding region primer. Sequence comparison between the complementary DNA and the gene revealed that the mouse sst5 gene contains 3 exons and 2 introns. The entire coding region was contained in exon 3. Two differently sized RACE products demonstrated alternate exon splicing of two untranslated exons in TtT-97 cells. A promoter fragment from -290/+48 linked to a luciferase reporter demonstrated 600- and 900-fold higher activity over a promoterless control in GH3 mammosomatotropes and TtT-97 thyrotropes, respectively, whereas a larger fragment extending to -6400 exhibited no additional promoter activity. Cloning of the sst5 gene will facilitate the mapping of basal and regulated responses at the transcriptional level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PEPTIDE HORMONE somatostatin (SS) is widely distributed in the central nervous system, pituitary gland, gastrointestinal tract, pancreas, kidney, and lymphopoetic tissue (1, 2). The native peptide circulates as two distinct forms containing 14 or 28 amino acids (SS-14, SS-28); both are potent inhibitors of basal and stimulated secretion from a wide variety of endocrine and exocrine target cells and exhibit antiproliferative effects (3). Somatostatin acts in an endocrine, autocrine, and paracrine fashion by binding to a family of transmembrane receptors, which are encoded by five related genes (sst1–5) located on different chromosomes (4, 5). These receptors are members of the G protein-coupled receptor family (GPCR) that contain a seven {alpha}-helix transmembrane structure and mediate ligand induced intracellular signaling by activation of multiple effector pathways (2). In general, within a particular target cell, the 5 sst subtypes have overlapping but characteristic patterns of expression that are subtype selective and tissue-specific with some species-specific variation (2).

The initial studies on the role of hypothalamic somatostatin elucidated its involvement in the physiological inhibition of pituitary GH and TSH in rodents and in humans (6, 7). While all five receptors are expressed in the adult rat pituitary (8), humans express sst4 only transiently during development, whereas the other four subtypes are present in the adult pituitary (9). Several groups have reported the colocalization of the various sst subtypes by in situ hybridization in all five of the major pituitary cells. For example, Day et al. have shown that sst5 messenger RNA (mRNA) to be present in 70% of rat somatotropes, 57% of thyrotropes, 38% of corticotropes, 33% of lactotropes, and 21% of gonadotropes; whereas sst2 expression occurred in 40% of somatotropes, 36% of thyrotropes, 26% of lactotropes, 3% of corticotropes, and 8% of gonadotropes (10). In this study using the rat model, the amount of mRNA per cell was higher for sst5 than sst2. In addition, two groups using a double immunostaining analysis have shown that sst2 and sst5 are the most widely distributed subtypes in the rat pituitary (11, 12). Within the human pituitary, sst2 and sst5 were found to be the most abundant subtypes by in situ hybridization (13). Finally, recent work in human fetal pituitary cultures using receptor-specific somatostatin analogs have suggested that sst5 is the principal subtype mediating GH and TSH suppression in human somatotropes and thyrotropes, while suppression of PRL is mediated by sst2 in normal cells and sst5 in prolactinomas (2, 14, 15). Together, these studies point to the abundance of sst2 and sst5 in rodent and human pituitaries and the critical role that they play in the suppression of GH, PRL, and TSH in normal physiology and disease.

Because the anterior pituitary contains multiple hormone-producing cells and a relatively low proportion of thyrotropes, we have begun to investigate the role of somatostatin on TSH suppression using the mouse TtT-97 tumor, which resemble normal thyrotrope cells by synthesizing both TSH subunits and exhibiting normal suppression of growth and TSH secretion by thyroid hormone (16, 17, 18). We have previously shown by both Northern blot and RT-PCR analyses that predominately sst5 and lesser amounts of sst1 mRNA are induced in these TtT-97 cells when animals were treated with thyroid hormone while sst2, sst3, and sst4 were undetectable (19). This was accompanied by a concomitant marked reduction in tumor growth (20). By contrast, under hypothyroid conditions, we did not detect any of the five somatostatin receptor subtypes by Northern blot or RT-PCR analyses (19). Furthermore, we showed that binding of 125I-labeled octreotide, a somatostatin analog with increased affinity for rodent sst2 and sst5, as well as 125I-labeled SS-28 binding, with highest affinity for sst5 (2, 21), were markedly increased on the surface of TtT-97 tumor slices from the T4-treated mice and could be competed with an excess of cold ligand. Thus, at least three independent lines of evidence show that thyroid hormone induces sst5 mRNA and protein expression in the TtT-97 thyrotropic tumor, and this phenomenon is coincident with the suppression of TSH secretion and the regression of tumor growth. To study the molecular mechanisms involved in the basal and regulated expression of this major somatostatin receptor subtype present in thyrotropes, we have cloned the mouse sst5 gene and have characterized its structure and promoter function in transiently transfected pituitary cells. While the coding region for this gene had been reported by several groups (23, 24, 25), the present study shows that the genomic structure is more complex, containing two 5' untranslated exons located several kb upstream, which can be differentially spliced to the coding region exon in TtT-97 thyrotropes. We have also localized strong promoter activity to a proximal region from -290 to +48 relative to the transcriptional start site in pituitary- derived thyrotropes and mammosomatotrope cell lines and identified consensus DNA binding elements that may have crucial importance in controlling the expression of the sst5 gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of the mouse sst5 gene
A mouse genomic library derived from a male BALB/c liver in bacteriophage {lambda}EMBL-3 SP6/T7 (CLONTECH Laboratories, Inc., Palo Alto, CA) was screened with a 1100 bp rat sst5 complementary DNA (cDNA) probe (kindly provided by Drs. J. Bruno and M. Berelowitz, SUNY-Stonybrook) by the plaque hybridization assay (26). The probe was radiolabeled with [{alpha}32P]-dCTP (ICI, 3000 Ci/mmol, 10 mCi/ml) by nick translation (27) to a specific activity of 5–7 x 108 cpm/ug. Approximately 1.25 x 106 plaques were plated onto a lawn of Escherichia coli K802 at 40,000 pfu/plate. Several strongly hybridizing plaques were isolated and rescreened through four rounds of hybridization until plaque pure. Recombinant DNA from the strongest hybridizing clone, termed {lambda}20A, was purified from a plate lysate using the Wizard phage purification kit (Promega Corp., Madison, WI). The insert was excised by digestion with SalI, which resulted in three fragments with a total size of 16 kb. Additional restriction digests of {lambda}20A were performed with BamHI, EcoRI, XhoI, and SalI alone and in combination; fragments were denatured, transferred to a 0.45 µm nitrocellulose sheet (Schleicher and Schull, Keene, NH) and probed with 32P-labeled 396 bp and 402 bp HincII rat sst5 cDNA fragments representing amino acids 1–266 as described (28) to establish a partial restriction map. Each of the SalI fragments of 6.1, 6.0, and 4.0 kb were subcloned into the SalI site of pGEM5Zf+ (Promega Corp.) and their borders sequenced by the dideoxynucleotide chain-terminiation technique by cycle-sequencing using plasmid primers adjacent to the cloning site (SP6, T7, M13F, and M13R) with EXCEL II DNA polymerase (Epicentre Technologies, Madison, WI). The complete coding region of both strands of the mouse sst5 sequence along with 1.4 kb upstream of the initiation codon and 350 bp downstream of the termination codon (GenBank no. AF030441) was determined by cycle sequencing using AmpliTaq DNA polymerase FS with an ABI Prism model 377 fluorescent sequencer (PE Applied Biosystems, Foster City, CA) with appropriate oligonucleotide primers (Life Technologies, Inc., Rockville, MD).

5' RACE and mapping of intron-exon junctions
To obtain the complete 5' untranslated sequence of msst5, we used a 5' RACE protocol using the Marathon cDNA amplification kit (CLONTECH Laboratories, Inc., Palo Alto, CA) with mouse TtT-97 thyrotropic tumor RNA from a T4-treated animal (19). Total RNA was isolated by the guanidinium isothiocyanate-CsCl method (29). PolyA+ RNA was isolated by affinity chromatography over two successive oligo-dT cellulose columns (type 7, Pharmacia Biotech, Inc., Piscataway, NJ) and treated with RNase-free DNase (Message Clean Kit, GenHunter, Nashville, TN). Briefly, 1 µg of polyA+ RNA was annealed to the modified oligo-dT primer, double-stranded cDNA synthesized by standard procedures (30) followed by treatment with T4 DNA polymerase to produce blunt termini and ligation to the marathon cDNA adaptor at both ends. The modified cDNA was diluted 1:50 with 10 mM Tris-Cl, pH 8, and 1 mM EDTA and PCR was performed with the 27 nt marathon adaptor primer (AP1) and a mouse sst5 coding region antisense gene-specific primer 5' TCC ACC CAG TCC CAC GGT GCA TAC CAA CA 3' complementary to codons 45–54. The amplification reaction was performed for a total of 30 cycles using a modified long distance PCR protocol with Takara Taq DNA polymerase (PanVera Corp., Madison, WI) mixed 1:1 with Taq-Start antibody (Promega Corp.). An aliquot of the reaction was size separated on a 2% agarose gel, fragments transferred to a Nytran membrane, and the blot was probed with a 32P-labeled more upstream coding region oligonucleotide 5' GGA GCC CGG GCG GTA TTA GT 3' corresponding to codons 34–40 to verify the specificity of the amplification. Two hybridizing species of 300 and 450 bp were detected. The remainder of the PCR reaction was electrophoresed through a preparative 2% agarose gel, fragments were excised, ligated to the T/A cloning vector, pCR2.1 (Invitrogen Corp., Carlesbad, CA), and screened for sst5 cDNA inserts by a colony screening procedure (31) using the radiolabeled oligonucleotide corresponding to codons 34–40, and a number of independent inserts from each size class were sequenced. We then used oligonucleotides within both ends of the exon 1 and exon 2 cDNA sequences for use in cycle sequencing of the 6.0 kb SalI genomic fragment in pGEM5Zf+ to establish the intron-exon junctions and to sequence the 5' flanking region.

Determination of the transcription start site
Because of the low abundance of sst5 mRNA in T4-treated TtT-97 thyrotropes, we employed an RT-PCR approach with a common antisense oligonucleotide within exon 3 from the longest 5' RACE cDNA along with several sense-strand oligonucleotides. The sequence of the exon 3 antisense oligonucleotide was 5' CCA GCT AGG TGT GGA AGC 3' (complementary to codons 7–12) and the sense strand oligonucleotides were within exon 1, 5' ATC CAG TGA GCG CTC TGC T 3' (E1S) or exon 2, 5' CTG TCC ACG GGA CAT GTG A 3' (E2S). Two additional sense strand oligonucleotides corresponding to sequences immediately upstream of the longest 5' RACE product consisted of 5' ATC TCC TCC ACC CTC TCC CT 3' (P1S, -42 to -23) and 5' TAG CCT GAG GGC GGG CGC (P2S, -17 to +1). As a positive RNA control for each PCR amplimer set, we constructed a pGEM7Zf+ plasmid containing msst5 promoter sequences from the SalI site at -290 to the XhoI site at +48 and fused it to the longest cDNA product at the unique XhoI site within exon 1. This construct, containing the promoter from -290 fused to exon1, exon2, and part of exon3 was linearized with EcoRI and sense strand RNA was synthesized in vitro with T7 RNA polymerase as described (32). RT-PCR was performed with each primer set as described (19) with 350 ng polyA+ RNA from T4-treated TtT-97 thyrotropes or 1 ng of the positive RNA control using random primers for the RT step and an annealing temperature of 58 C during the 35 cycle PCR step. Products of each reaction and a yeast transfer RNA control (2.5 µg) were electrophoresed through a 1.25% agarose gel, denatured, transferred to a Nytran membrane as described (28), and probed for amplified sst5 sequences with a 32P-labeled sense strand oligonucleotide 5' TGT GCT CTG GCA TCC TGA ACC TG 3' containing sequences just upstream of the translation initiation site within exon 3.

Plasmid constructions for transfection studies
A promoterless luciferase plasmid, pA3LUC (33) was modified to contain additional cloning sites to facilitate subcloning of msst5 promoter fragments. The vector was cleaved with KpnI and HindIII, gel purified, and ligated to a duplex oligonucleotide containing overlapping KpnI and HindIII termini. The sense strand oligonucleotide was 5' CAC ACT AGT CTC TGC AGG ACC ATG GTA GTC GAC TCA 3' and the antisense strand was 5' AGC TTG AGT CGA CTA CCA TGG TCC TGC AGA GAC TAG TGT GGT AC 3'. The resultant plasmid termed pA3LUC+ contained unique recognition sites for SpeI, PstI, NcoI, and SalI, in addition to the KpnI, SmaI, and HindIII sites in the parent vector. Initial promoter fragments contained sequences with varying lengths of 5' DNA located at the following distances upstream of the translation initiation site: these were 5.6 kb (SalI), 4.65 kb (NdeI), 3.8 kb (HindIII), 3.1 kb (XhoI), and 1.3 kb (BamHI). The 3' extent of all of these fragments was a unique SpeI site located 61 bp upstream of the initiation codon. A 5.6 kb SalI to SpeI fragment was excised from the 6.0 kb SalI genomic fragment in pGEM5 Zf+ by digestion with SpeI (one site contained in the vector) and ligated into pA3LUC+ at the SpeI site; both forward and reverse orientations of this construct were prepared. A 3.8 kb HindIII to HindIII fragment (latter site in vector) was excised from the 5.6 kb SalI to SpeI fragment in pA3LUC+ and subcloned into the unique HindIII site of pA3LUC resulting in a 3.8-kb HindIII to SpeI genomic fragment in the luciferase vector. A similar strategy was used to subclone a 3.1 kb XhoI to SpeI fragment. Isolated 4.65 kb NdeI to SpeI and 1.36 kb BamHI to SpeI fragments were end-filled with reverse transcriptase and all four dNTPs and ligated to SmaI linearized pA3LUC+. The borders of all of these constructs were verified by DNA sequencing.

A smaller upstream SalI to XhoI fragment from -290 to +48 was excised from the 5.6 kb SalI to SpeI fragment in pA3LUC+ and subcloned in the forward orientation into the SalI site of pA3LUC+; only the upsteam SalI site was preserved in this construct. The 6.1 kb SalI genomic fragment from approximately -6400 kb to -290 was isolated and subcloned in the forward orientation at the unique SalI site of the -290 to +48 SalI to XhoI fragment in pA3LUC+. The sequence at the -290 SalI junction of this large promoter construct matched the sequence upstream of position -290 within the original recombinant phage DNA.

Transient transfection in pituitary cells
Transient transfection assays in TtT-97 thyrotropic tumor cells have been previously outlined (18). Briefly, 20 µg of the various mouse sst5 promoter-luciferase plasmids and 2 µg pCMVbgal as an internal control for transfection efficiency were cotransfected by electroporation into 5–10 million TtT-97 cells. Cells were incubated in 4 ml of DMEM supplemented with charcoal stripped 10% FCS (Life Technologies, Inc.) at 37 C for 18 h. Transfection of monkey kidney CV-1 cells by the calcium phosphate method were previously described (34) and rat pituitary-derived GH3 or GH4T2 mammosomatotrope cells by electroporation were described previously (35). GH3 and GH4T2 cells each express both GH and PRL although at different relative levels. GH4T2 cells were developed by passaging GH4 cells in rats and cell lines established from the resulting tumors (gift from Dr. A. Gutierrez-Hartmann). Cells were harvested, subjected to freeze-thaw extraction, and assayed for luciferase and ß-galactosidase activity as previously described (36). Luciferase activity was measured in a Monolight 2010 luminometer from duplicate aliquots of freeze-thaw cytoplasmic lysates (18) from the cells, while ß-galactosidase activity was measured using a colorimetric assay (37) and were compared with a standard curve of enzymatic activity. Light units were normalized to the ß-galactosidase activity and were corrected to the activity of an RSV-luciferase construct transfected in parallel. Statistically significant differences were tested by one-way ANOVA.

Animal treatment
Studies on LAF1 mice bearing TtT-97 thyrotropic tumors were conducted with the highest standards of humane animal care in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The protocols were approved by the Committee on Animal Care and Use of the University of Colorado Health Sciences Center (Denver, CO).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of a genomic clone encoding the mouse sst5 gene
We screened a mouse genomic library for sequences that positively hybridized with a full-length rat sst5 cDNA insert. From a total of 1.25 x 106 phage, nine plaques hybridized with the 1100 bp rat sst5 cDNA insert after the first round of screening. These were replated and rescreened through successive second, third, and fourth rounds of purification until two recombinants were plaque pure. The strongest hybridizing clone was chosen for further analysis. DNA was prepared from plate lysates and digested with SalI, a site that borders both ends of the BamHI/Sau 3A insert within the vector. This resulted in three genomic fragments with sizes of 6.1, 6.0, and 4.0 that were individually subcloned into pGEM5Zf+. The two smaller fragments hybridized with a rat sst5 probe representing the amino-terminal 2/3 of the coding region. Sequence determination of the borders of each of the genomic fragments allowed an unambiguous ordering of the fragments within the vector. Each SalI fragment was further digested with a variety of restriction enzymes alone or in combination, resulting in a partial restriction map as is shown in Fig. 1AGo. DNA sequencing revealed an open reading frame of 362 amino acids corresponding to the complete coding region of mouse sst5 that was not interrupted by introns as has been reported for all the other sst subtype genes except mouse sst2 (38). Genetic database comparisons with the known rat and human sst5 coding regions demonstrated a high degree of homology in primary protein structure. There were 351/363 (96.7%) of amino acids that were identical when compared with the rat and 300/364 (82.4%) identical with the human receptor. The coding region and 1392 bp upstream of the initiation codon and 353 bp downstream of the termination codon have been deposited in GenBank as accession number AF030441. The nucleotide sequence of the coding region agreed exactly with the sequence determined by Baumeister et al., 1998, GenBank AF035777 (25). However, the latter group assigned an additional 23 amino acids to the amino terminus of the open reading frame for a total of 385 amino acids.



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  		Figure 1. Restriction map of the mouse sst5 gene. A 16-kb mouse sst5 genomic fragment was excised from {lambda}20A. A, Schematic of several restriction enzyme sites in relation to the lambda phage arms. The coding region is shown as a gray box. The inset was excised from the vector with SalI, and the location of the three resultant fragments are shown by brackets below the linear map. B, Size and relative location of genomic fragments that were fused to firefly luciferase used to test for promoter activity in pituitary cells.

 
Mapping sequences mediating promoter activity
Because the human sst5 gene was reported to be devoid of introns in both the coding and 5' untranslated regions (39), we initially used various fragments of DNA directly upstream of the translation initiation site to test for promoter activity in transient transfection studies into sst5 expressing rodent thyrotrope and mammosomatotrope-derived cells. We subcloned genomic fragments of 5.6 kb (SalI), 4.65 kb (NdeI), 3.8 kb (HindIII), 3.1 kb (XhoI), and 1.3 kb (BamHI) upstream of the coding region (see Fig. 1AGo) and fused them to firefly luciferase in the pA3LUC+ vector. The 3' end of all constructs terminated at a unique SpeI site 61 bp upstream of the initiation codon and are shown schematically in Fig. 1BGo.

To test for promoter activity, each fragment was transiently transfected into GH4 and GH3 rat mammosomatotrope cell lines (Fig. 2Go) and luciferase activity, normalized to the enzymatic activity of a cotransfected internal CMV-ßgal plasmid, was obtained for three or more independent transfections. In addition, the largest 5.6-kb fragment was tested in the reverse orientation. The results show that only the largest 5.6-kb fragment demonstrated substantial promoter activity that was 300- to 500-fold over that obtained by the promoterless construct in these cells. In addition, it demonstrated significantly higher activity when compared with each of the shorter fragments tested (P < 0.001). The next largest fragment of 4.65 kb showed a 6.5-fold drop in promoter activity in GH3 cells with a further 2- to 4-fold drop in activity with the 3.8-, 3.1-, and 1.3-kb genomic fragments. Furthermore, the activity of the 5.6-kb fragment was orientation dependent because it was at least 30-fold less active in both GH4 and GH3 cells when tested in the reverse orientation (P < 0.001). The 5.6-kb and 1.3-kb genomic fragments were also tested in dispersed TtT-97 thyrotropic tumor cells (Fig. 2Go). The largest fragment again showed substantial promoter activity that was about 25-fold higher than the smaller 1.3-kb fragment. In contrast to the findings in pituitary derived cells, the 5.6-kb fragment had similar activity to the 3.1 and about half the activity of 1.3-kb fragments in CV-1 monkey kidney cells (Fig. 2Go). This activity was still 50–100 fold greater than the promoterless plasmid and exhibited orientation dependence for the 5.6-kb fragment. These results demonstrate that strong promoter activity maps to the region between 5.6 kb and 4.65 kb upstream of the translation start site and cell-specific differences were observed in pituitary vs. nonpituitary cells. This suggests that either an enhancer element is contained far upstream of the coding region or that the authentic promoter region is located upstream of one or more 5' untranslated exon(s) separated from the coding region by intervening sequences as has been shown for the rat sst3 gene (40) and the mouse sst2 gene (41).



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  		Figure 2. Promoter activity of genomic fragments in pituitary somatotropes, thyrotropes, and nonpituitary cells. The fragments depicted in Fig. 1BGo were fused to a luciferase reporter, and 20 µg of the expression vector were transiently transfected into GH4 or GH3 mammosomatotrope cell lines, dispersed primary TtT-97 thyrotrope tumor cells, or CV-1 monkey kidney cells. Promoter activity is shown for each construct as fold increase relative to the activity of the promoterless pA3LUC vector. In the TtT-97 cells, activity is expressed in light units normalized to an internal CMV-ßgal construct. The largest 5.6-kb fragment was inserted into the expression vector in both forward (F) and reverse (R) orientation. The error bars show the SEM for duplicate aliquots from at least three independent experiments. Asterisks denote statistically significant differences (P < 0.01) using one-way ANOVA.

 
Structure of the 5' untranslated region and intron-exon organization of the msst5 gene
To determine the extent of the 5' untranslated region, we performed a 5' RACE (rapid amplification of cDNA ends) analysis using polyA+ RNA from sst5 expressing TtT-97 thyrotropes from T4 treated mice as described in Materials and Methods. Two differently sized cDNAs of 450 and 300 bp were identified by Southern blot analysis with an internal exon 3 specific probe, PCR products were cloned into the PCR2.1 vector, and several independent clones from each size class were completely sequenced. Each product contained the sst5 gene specific oligonucleotide at one end and the adaptor primer sequence at the other end that were used in the amplification reaction. Sequences from each size class differed from each other at their 5' termini by no more than about 10–15 bp. We then compared the structure of each cDNA with the gene sequence and found that they both diverged from the genomic sequence 35 bp upstream of the translation initiation site. The smaller sized cDNAs differed from the larger one by the lack of an internal 154-bp sequence. This was due to an additional exon (exon 2) that is alternatively spliced in TtT-97 RNA. The nucleotide sequence of each cDNA had strong homology with the published rat sst5 cDNA (GenBank no. LO4535.1) (21). There were only five nucleotide differences within the first 60 bp of the mouse when compared with the rat sequence. A schematic showing the cDNA structure and a partial comparison with the rat cDNA is shown in the top of Fig. 3Go. The homology with the rat sequence extended throughout the first exon of 114 bp and then diverged. Interestingly, the next 24 bp in the rat 5' UT significantly differs in sequence from the mouse and suggests the presence of a second, nonhomologous exon in the rat gene. Using oligonucleotides to just within the borders of exons 1, 2, and 3, we determined the sequence of the intron-exon boundaries from our genomic clone and of the probable 5' flanking DNA. The distance between each of the exons within the gene was determined by PCR using pairs of exon-specific amplimers. The 5' border of each intron began with a GT dinucleotide and the 3' border ended with the dinucleotide AG, a feature characteristic of most eukaryotic introns (42). The structural organization of the mouse sst5 gene is depicted schematically on the bottom of Fig. 3Go.



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  		Figure 3. Structure of sst5 transcripts and the genomic organization of the sst5 gene. Two distinct splice variants of mouse sst5 transcripts are shown in panel A based on the sequence of the major 5' RACE cDNA products. Exon1 is a gray box and is spliced to either exon 2 (white box) or to exon 3 (black box). The nucleotide sequence of the first 42 bp of exon 1 of the mouse (m) sst5 cDNA (top) is compared with the published rat (r) sst5 cDNA (bottom) to show similarities between the sequences with vertical lines showing identity between the sequences. B, Schematic of the gene organization of the 3 exons and 2 introns. The coding region within exon 3 is intronless. The large 5.6-kb genomic SalI to SpeI fragment used in transfection studies is shown with a bracket.

 
Mapping the transcriptional start site and mouse sst5 gene sequence
To see if the 5' extent of the RACE products represented the transcriptional start site, we performed a series of RT-PCR reactions with a common antisense strand oligonucleotide contained in the coding portion of exon 3 and a series of sense strand oligonucleotides within exon 1, exon 2, or just upstream of the longest cDNA products obtained by 5' RACE. A schematic describing this strategy is shown in Fig. 4AGo. For use as a positive RNA template, we constructed a pGEM7 plasmid containing promoter sequences from -290 to +48 fused to the longest 5' RACE product at the unique XhoI site at position +48. The plasmid was linearized with EcoRI and a sense strand RNA was synthesized in vitro with T7 RNA polymerase. This resulted in an RNA containing the -290 promoter fused to the two 5' untranslated exons (1, 2) and to the coding region exon 3. This control was designed to test that all the primer sets were capable of amplifying the predicted product starting with RNA. The results of this analysis shown as a Southern blot probed with an internal exon 3 oligonucleotide is displayed in Fig. 4BGo. Using an E1S and E3A primer set, RT-PCR amplification of TtT-97 RNA resulted in a strong 320 bp transcript (lane 2) and a much weaker 168 bp transcript. These represent amplification of RNA species containing exons 1, 2, and 3 (E1,2,3) as well as the shorter E1,3 species respectively, and correlate with the two RACE products described above. This suggests that the shorter transcript, which lacks exon 2, is much less abundant in TtT-97 RNA than the larger species. The control RNA only yielded the larger transcript as expected (lane 4), whereas the yeast RNA control was negative (lane 3). For the E2S and E3A primers, a single 207 bp fragment was present in the TtT-97 (lane 2) and control RNA (lane 4). Both spliced products contributed to this band in the thyrotrope cells. In contrast, primer sets P1S and E3A or P2S and E3A, failed to amplify an RNA in the TtT-97 sample (lane 2), whereas the appropriate sized band of 379 bp or 353 bp were present with the control RNA (lane 4). This shows that TtT-97 RNA could not serve as a template for either of the two sense strand primers located just upstream of the longest 5' RACE cDNA and verifies that the start site is located 114-bp upstream of the beginning of the first intron and 290 bp downstream of a SalI site that was included in the genomic fragment exhibiting the strongest promoter activity (Fig. 2Go).



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  		Figure 4. Mapping the transcriptional start site by RT-PCR. The top of panel A shows a schematic of the 5' flanking region (promoter) fused to both alternatively spliced sst5 transcripts found in TtT-97 thyrotropic tumors. Above and below the structure are shown the successive sense strand or the unique antisense strand primers used for the RT-PCR studies. The top species was contructed in vitro as a sense strand RNA containing the 290-bp promoter fused to exons 1, 2, and 3 and served as a positive control for each amplimer set. B, Results of the amplification reaction with each primer set after products were electrophoresed through an agarose gel, transferred to a Nytran membrane, and probed with a 32P-labeled oligonucleotide just within exon 3. Arrows show the location and sizes of the hybridized fragments. Lane 1, 32P-labeled pBR322 DNA cleaved with HpaII used as size standards; lane 2, TtT-97 poly A+ RNA; lane 3, yeast transfer RNA; and lane 4, sense strand positive control RNA.

 
Nucleotide sequence of the msst5 gene
The nucleotide sequence of 870 bp of the 5' flanking region, the two 5' untranslated exons, the coding region with the predicted primary amino acid sequence, and the intron-exon junctions are shown in Fig. 5Go. The 5' untranslated region is contained within exons 1, 2, and 3 and comprises either 301 bp or 149 bp for the two alternately spliced transcripts. No upstream in-frame ATG sequences exist in either of these transcripts. In this study, we show 353 bp of the 3' untranslated region. Because the full-length RNA transcript is about 2.6 kb (19), there are about 900 bp of additional 3' untranslated region that have not been characterized. The promoter lacks a TATAA sequence but does contain a CCAAT box at position -166. In addition, several putative consensus sites for transcription factors are present including half sites for steroid and thyroid hormone receptors, as well as GATA, AP-1, and Ets sites; the location of which are shown in Fig. 5Go. While no consensus Pit-1 sites are found, sites for the transcription factors Pitx1 and Isl-1, which have been described in pituitary cells, are present at positions -61 and -181, respectively.




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  		Figure 5. Nucleotide sequence of the mouse sst5 gene. The nucleotide sequence of the sense strand is shown beginning at position -870. Numbers to the left indicate positions relative to the major transcriptional start site. The 5' flanking region is shown in italics and putative consensus recognition sites for various transcription factors are underlined, exons are shown in boldface, and intron sequences are shown in small letters. The predicted amino acid sequence for sst5 is shown above each codon with the initiation and termination codons double underlined.

 
Mapping of promoter activity in pituitary cells
Earlier we presented data to show that strong promoter activity in transfected pituitary thyrotropes and mammosomatotrope cells mapped to the largest 5.6 kb genomic fragment bordered by SalI and SpeI sites and that promoter activity declined considerably with the next smallest fragment at the NdeI site. Due to the revised genomic organization presented in Fig. 3Go, we show that the SalI site is located at position -290 relative to the transcriptional start site and that the SpeI site maps near the 3' end of the second intron, 61 bp upstream of the translation initiation site. We therefore tested the promoter activity of a 338 bp fragment extending from -290 bp to +48, the latter site located within the first exon to eliminate the contribution of intron sequences. As is shown in Fig. 6Go, the shorter -290/+48 fragment demonstrated strong promoter activity of 600-fold above the promoterless vector in transfected GH3 mammosomatotropes and 900-fold in TtT-97 thyrotropic tumor cells. These values are significantly larger than the value obtained for the 5.6 kb SalI to SpeI fragment in GH3 cells (P = 0.001) and demonstrated that the smaller promoter fragment accounted for all the transcriptional activity. The lower values of the larger genomic piece may reflect some inhibitory sequences present in intron sequences and may be partially accounted by the fact that about half of the number of molecules were transfected into the cells when an equal mass of each plasmid were used.



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  		Figure 6. Promoter activity of 5' flanking segments of the sst5 gene. GH3 and TtT-97 cells were transfected as described in Materials and Methods with various sst5 genomic fragments fused to a luciferase reporter to test for promoter activity. The results are expressed as fold activity for duplicate aliquots from at least three independent transfections ± SEM relative to the activity of the promoterless pA3LUC plasmid. The fragments are the 5.6-kb SalI to SpeI genomic fragment in the forward orientation (F) (see Fig. 1Go), the -290 to +48 (SalI to XhoI), and the -6400 to +48 region (most upstream end of genomic clone {lambda}20 to XhoI).

 
We then tested whether additional promoter sequences resulted in higher promoter activity by testing a construct containing an additional 6.1-kb fused in the forward orientation to the -290 to +48 construct. As shown in transfected GH3 cells (Fig. 6Go), this -6400/+48 fragment demonstrated less promoter activity than the -290/+48 fragment. This may reflect suppressor elements contained between -290 and -6400 and/or may be accounted for the fact that about half the moles were present for this construct when compared with the smaller fragment. When equal moles of each fragment were directly compared, no significant differences in promoter activity were found (data not shown). Thus, we have more precisely localized the promoter activity to the region of the sst5 gene between -290 and +48 in pituitary derived cells. This region contains putative binding sites for several transcription factors including Sp1, GATA, and the more cell restricted factors found in pituitary cells, Pitx1, and Isl-1. Future studies will determine if these factors bind and functionally interact with cognate sequences within this promoter.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have isolated and characterized the mouse somatostatin type 5 receptor gene and have shown that it consists of 3 exons and 2 introns. While the coding region for this receptor is devoid of introns, the 5' untranslated region is comprised of two upstream, optionally spliced exons of 114 bp and 152 bp, which are separated from the remaining 35 bp of 5' UT and the protein open reading frame within exon 3 by introns of approximately 2 and 3 kb. The predicted amino acid sequence of the sst5 protein contains 362 amino acids. This is different than the 385 amino acids described by another group (25). However, the additional 23 amino acids at the amino terminus of that study (25) may be artefactual because the assigned ATG initiation codon lies within intron sequences upstream of the intron2-exon3 splice junction. Similar to the recently reported structures of the mouse sst2 (41) and rat sst3 genes (40), the mouse sst5 gene contains intervening sequences in the 5' untranslated region, whereas the coding region is intronless. In excess of 90% of mammalian G protein-coupled receptors (GPCR), gene sequences contain no introns in their coding regions (43). In a recent survey of GenBank sequences, among those mammalian GPCR genes lacking introns in their open reading frames, 18% possess introns in their 5' untranslated region, 33% contain no introns, none contain introns in their 3' untranslated region, and the rest have not been completely characterized (43). In contrast, all the 60 characterized GPCR genes in C. elegans contain introns in their coding region. The reasons for these curious species-specific differences are unknown. Intronless genes might be more efficiently transcribed, although in Drosophila, introns have been shown to increase gene expression levels (44). However, genes without introns are not subject to differential or aberrant splicing, and thus may result in higher fidelity. Introns within the 5' UT may allow for differential use of alternate promoters permitting distinct regulatory patterns of gene expression in a tissue-specific manner. In fact, a recent report using RT-PCR inferred that the mouse sst2 gene has multiple promoters that are differentially active in different tissues, and that presumably might have independent modes of regulation (41). However, a direct examination of promoter function was not performed in this study. In contrast, the cloned human sst2 gene appears to be intronless even within the 5' UT, and demonstrates promoter activity in transfected neuroblastoma cells, with cis-acting elements present for the transcription factors SEF-2 and MIBP1 located within 100 bp upstream of the translation initiation site and 5–22 bp from the transcriptional start site (45, 46). In contrast, another report characterizing the human sst2 gene, localized strong promoter activity in T47D cells to sequences located between 5.3 and 3.8 kb upstream of the coding region (47). A region with strong promoter activity several kb upstream is similar to our present studies on the mouse sst5 gene and suggests that perhaps splicing of upstream exons, or multiple promoters exist for many members of the sst gene family. In the current study, it doesn’t appear that an additional promoter occurs in the sequence upstream of exon 2 because much lower promoter activity was found in pituitary cells with all of the constructs whose 5' termini were downstream of exon 1. However, the promoter activity was not completely abolished in the shorter constructs. Future studies testing these promoter fragments in a variety of cells along with RT-PCR studies will be necessary to test for the presence of alternate promoters in the sst5 gene that are used in other cell types.

In this study, we have also shown that at least two forms of sst5 transcripts can be detected in TtT-97 thyrotrope derived cells although the splicing of E1 to E2 to E3 predominates over transcripts that result from splicing of E1 directly to E3 that lack E2. The presence of alternate 5' untranslated regions of 301 or 149 bp may allow for differential stability of the processed transcript or may have an impact on translation. We cannot rule out, however, that other tissues may predominantly express the smaller form. Future RT-PCR studies using RNA isolated from other sst5 expressing cells using our amplimer sets can test whether this occurs in vivo. Additionally, coexpressing both of the sst5 forms and including an epitope tag can test whether there are differences in RNA stability or translational efficiency.

Recently, Brinkmeier and Camper have localized the mouse sst5 gene near a quantitative trait locus (QTL) on Chr 17 that affects early growth, and that is located 2 cM proximal to the marker D17Mit46 (5). Creation of null alleles by gene targeting will be important for determining the role of the sst5 receptor in growth and thyroid status because it appears to play a major role in controlling somatostatin mediated suppression of GH and TSH in humans (14) and in other mammals (2). Because thyroid hormone also acts to suppress pituitary TSH, we have begun to investigate the potential linkage between somatostatinergic and T3/T4-mediated effects. In this regard, we have shown in hypothyroid mice treated with excessive (19) or physiological levels of thyroid hormones (Woodmansee, W. W., D. F. Gordon, J. M. Dowding, B. Stolz, R. V. Lloyd, R. A. James, W. M. Wood, and E. C. Ridgway, manuscript submitted), that there is an induction of sst5 mRNA and protein expression in TtT-97 tumors from the undetectable levels seen in hypothyoidism. This suggests that a transcriptional mechanism may be in place to regulate biosynthesis of this receptor subtype either directly or indirectly by thyroid hormone. Interestingly, a consensus thyroid hormone response element (TRE) half site is present beginning at position -140 in the sst5 promoter that may be involved in mediating this response.

The TtT-97 tumor is a pure thyrotrope model that exhibits both an antisecretory event (TSH suppression) and an antiproliferative effect (tumor regression), which are both mediated by thyroid hormone, and that correlate with sst5 expression. The determination of the cis-acting sequences and trans-acting factors involved in the regulation of the sst5 gene is essential to elucidate the molecular mechanisms controlling its function in the pituitary and has implications for normal physiology and pathology. It is interesting to speculate that the pituitary-restricted transcription factors Pitx1 (48) or the related Pitx2 factor (48A ), which are bicoid-related homeoproteins, and the LIM homeodomain factor, Isl-1 (49), for which consensus binding sites exist within the functionally important -290/+48 promoter region of sst5, may be involved in regulation of its expression in pituitary cells. In addition, the sst5 gene is expressed in other tissues including the endocrine pancreas. In the rodent, sst5 has been implicated as the receptor responsible for regulation of insulin secretion from pancreatic islet cells by somatostatin (50). Consensus binding sites for factors found primarily in the pancreas, PTF1 (51), a basic helix loop helix factor, and Isl-1, originally cloned from islet cells (49), are also found in the -290/+48 promoter region. Future studies, using cotransfected transcription factors and mutagenesis of selected sequences, will be performed to test their role on the functional activity of the promoter.

In summary, we have isolated the mouse sst5 gene, characterized its genomic organization, and mapped high levels of promoter activity to a 300-bp region adjacent to the transcriptional start site. These studies lay the groundwork for determining the molecular mechanisms involved in its transcriptional regulation in sst5 expressing cells.


   Acknowledgments
 
We thank Heidi Knauf for assistance with the genomic screening, Drs. J. Bruno and M. Berelowitz (SUNY-Stonybrook) for the rat SST5 cDNA, and the DNA Sequencing and Analysis Core Facility of the University of Colorado Cancer Center (NIH/NCI Cancer Core Support Grant, CA-46934) for assistance with automated sequencing. We would also like to thank Dr. J. Tentler and A. Gutierrez-Hartmann for providing the GH4T2 cell line.


   Footnotes
 
1 Support for this work was provided by NIH Grants RO1-DK-36843, RO1-DK-47407, and RO1-CA-47411 (to E.C.R.). Partial support was also provided by an Endocrine Fellowship grant (to W.W.W.) from the American Thyroid Association. Back

Received June 1, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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