(Received for publication, August 8, 1995; and in revised form, September 1, 1995)
From the
Several cDNA clones encoding the human neuropeptide Y-Y1
receptor have been isolated that contain differing sequences at their
5`-ends. The divergence occurs at a splice junction in the
5`-untranslated region, suggesting that at least three forms of the
neuropeptide Y-Y1 receptor transcript are generated by alternative
splicing at this site. Genomic clones have been isolated that encompass
the alternatively spliced 5`-exons. The exons are found 6.4, 18.4, and
23.9 kilobases upstream of exon 2. In the corresponding promoter
regions of the various exons, possible response elements for the
glucocorticoid receptor, as well as potential binding sites for the
AP-1, AP-2, and NF-B transcription factors are found. Analysis of
NPY-Y1 transcripts in various cell types demonstrates the
tissue-specific activation of the three promoters.
Neuropeptide Y (NPY) ()is an abundant neuropeptide
that mediates a wide range of biological actions including the
regulation of appetite, cardiovascular activity, and endocrine
secretion. NPY receptors belong to the family of G protein-coupled
receptors, and several receptor subtypes have been classified on the
basis of their pharmacology (reviewed in (1) ). The NPY-Y1 and
NPY-Y2 receptor subtypes can be distinguished by their ability to bind
various analogs and fragments of NPY. The NPY-Y1 receptor selectively
binds an analog of NPY,
[Leu
,Pro
]NPY, while the NPY-Y2
receptor has higher affinity for the NPY (13-36)
fragment(2, 3) .
The NPY-Y1 receptor is expressed in many tissues including the brain, heart, placenta, spleen, small intestine, kidney, testis, and aortic smooth muscle(4, 5) . In the cardiovascular system, the NPY-Y1 receptor mediates both direct vasoconstriction and the potentiation of other pressor agents such as norepinephrine(6) . Administration of Y1 selective agonists also affects appetite and the secretion of luteinizing hormone, suggesting involvement of the NPY-Y1 receptor(7, 8) . NPY is considered to be the most potent endogenous orexigenic agent known with infusions of NPY into the paraventricular nucleus of rats inducing a sizable and long lasting increase in food intake(9) .
DNA sequences encoding the NPY-Y1 receptor have been cloned from several species including human, rat, mouse, and Xenopus(10, 11, 12, 13) . The human cDNA encodes a protein 384 amino acids in length that is preceded by approximately 200 base pairs (bp) of 5`-untranslated region (5`-UTR) sequence. Analysis of the genomic structure has determined the presence of a 6.4-kb intron within the 5`-UTR as well as a small intron within the coding region. The gene for the receptor has been localized to chromosome 4q(31.3-32)(14) .
During the analysis of NPY-Y1 receptor cDNAs from a testis cDNA library, a cDNA clone was identified that differed from the previously determined NPY-Y1 receptor sequence at the 5`-end of the clone. The two sequences diverged at the point where exon 1 splices onto exon 2. This suggests that two exons are alternatively spliced at this site. Although this has no effect on the protein sequence of the receptor, it does have consequences for the regulation of the gene, as transcription may be under the control of multiple promoters.
To determine whether alternative promoters are used in the transcription of the NPY-Y1 receptor gene, cDNA clones were isolated from a range of different cDNA libraries, and the 5` sequences of NPY-Y1 mRNA were examined by 5`-rapid amplification of cDNA ends (RACE). Alternate 5`-exons were identified in the structure of the NPY-Y1 receptor gene, and the selective activation of the different promoters for the NPY-Y1 receptor was investigated by measuring the expression of the various NPY-Y1 transcripts in different cell types.
Figure 1:
Schematic representation of the human
NPY-Y1 receptor gene. A partial BamHI (B), HindIII (H), and EcoRI (E)
restriction endonuclease map of the NPY-Y1 gene is shown. The lines represent intronic sequence, and the closed boxes represent exons encoding 5`-or 3`-UTR sequence. The open boxes denote the coding region of the gene. Also shown are the genomic
clones that span the gene.
Figure 2: Exon/intron structure and splice sites in the 5`-UTR of the human NPY-Y1 receptor gene. Open boxes represent untranslated sequence, and closed boxes represent the coding region of the gene.
Figure 3: 5`-Exon and flanking sequences of the human NPY-Y1 gene. The 5`-flanking sequences of exon 1A, 1B, and 1C are shown along with the exon sequences. The exons are underlined, and numbering begins at the major transcription start site for each of the exons. Minor transcription start sites are marked with asterisks. The potential recognition sequences for various transcription factors are boxed.
Figure 4: Transcription start sites of the 5`-exons determined by primer extension. cDNA was reverse transcribed from SK-N-MC mRNA using radioactively labeled primers, each designed to anneal to a different 5`-exon sequence (lanes 3, 6, and 9). Also shown are negative controls that had either reverse transcriptase (lanes 1, 4, and 7) or RNA (lanes 2, 5, and 8) omitted from the reaction. A DNA sequencing reaction was run next to the reactions to enable the size of the products to be determined.
Promoter A lacks a typical TATA and CCAAT box. However, it contains a potential cyclic AMP response element, 5`-CGACGTCA-3`, at position -92 and an AP-1 binding site, 5`-TGAATCA-3`, at position -763(17, 18) . Also present is a putative binding site for octamer-binding proteins (Oct-1), 5`-ATTTGCAT-3`, at position -889(19) .
Promoter B also lacks the
canonical TATA and CCAAT motifs and has a high G+C content
(approximately 70%) in the 200 bp preceding the transcription start
site. Two possible binding sites, 5`-CCGCGGGC-3`, for the transcription
factor AP-2 are found at positions -49 and -110 (20) . A glucocorticoid response element, 5`-TGTTCT-3`, is
present at position -585 (21) and a putative AP-1 binding
site, 5`-TGAATAA-3`, at position -744(18) . A recognition
site for NF-B is present at position -914(22) .
The only obvious transcription factor binding site in promoter C is a potential response element for the glucocorticoid receptor at position -357(21) . The promoter also lacks a TATA and/or CCAAT box.
Figure 5: Tissue-specific expression of the 5`-exons. RNA from artery (lanes 1, 5, and 9) and kidney (lanes 2, 6, and 10) tissue and the SK-N-MC cell line (lanes 3, 7, and 11) was reverse transcribed and then amplified using one 5`-exon-specific primer and one primer for exon 2. Lanes 1-4 used a primer specific for exon 1A, lanes 5-8 have the exon 1B-specific primer, and lanes 9-12 contain the exon 1C-specific primer. PCR products were detected by hybridization to an internal oligonucleotide (Y1#2). Lanes 13, 14, and 15 show part of the NPY-Y1 coding region amplified from the three cell types, demonstrating that approximately equal levels of NPY-Y1 transcript are present. Lanes 4, 8, and 12 are negative controls containing no template.
NPY-Y1 transcripts containing exon 1A and exon 1B could be detected in both the kidney and arterial tissues and the SK-N-MC cell line. Exon 1C could be amplified from the SK-N-MC cell line and to a much lesser extent from kidney tissue. Transcripts containing exon 1C could not be detected in arterial RNA.
The genes encoding G protein-coupled receptors comprise a
very large gene family. Although many members of this family have been
found to lack introns, many others have a non-contiguous structure,
including a growing number of genes that have been found to contain
introns within the 5`-UTR. Receptor genes displaying alternative
splicing within the 5`-UTR include the porcine muscarinic acetylcholine
receptor, the rat endothelin ET receptor, the human
interleukin 8 IL8RB receptor, and the human thromboxane receptor (23, 24, 25, 26) .
Analysis of NPY-Y1 transcripts expressed in the SK-N-MC neuroblastoma cell line shows that at least three exons are alternatively spliced onto the 5`-end of the NPY-Y1 receptor mRNA. The murine NPY-Y1 receptor gene has also been cloned and, like the human gene, has a 6-kb intron situated approximately 150 bp upstream of the start codon(12) . Primer extension of the murine NPY-Y1 mRNA, to determine the transcription-initiation site, revealed the presence of five transcripts of various sizes. This was interpreted to be the result of multiple transcription-initiation sites within the same exon sequence. A limitation of this method is that the sequences of the transcripts are not known, and it cannot be distinguished whether the different-sized transcripts are a continuation of the same exon sequence or the result of a splicing event. In the light of this work on the human NPY-Y1 gene, it is very likely that at least some of the multiple transcription-initiation sites found in the mouse gene are also the result of different 5`-exons being spliced onto the splice acceptor site within the 5`-UTR.
Alternative splicing of sequences within the 5`-UTR has been demonstrated to regulate receptor expression at the translational level (27) . For example, the presence of open reading frames within the 5`-UTR can result in the inefficient initiation of translation and suppression of protein expression(28) . The human NPY-Y1 receptor gene contains a short open reading frame in the 5`-UTR situated on exon 2. In addition, exon 1C also contains a short open reading frame; however, no start codons are present in either exon 1A or 1B. The differential use of these 5`-exons may be a mechanism for translational regulation of the NPY-Y1 receptor protein.
The selective activation of the promoters for the NPY-Y1 receptor was investigated by analyzing the relative abundance of the three different forms of Y1 transcript in various tissues. Reverse transcriptase-PCR demonstrated that exon 1C was expressed in the SK-N-MC cell line, to a lesser extent in kidney, and not at all in artery, indicating that promoter C is activated in a tissue-specific manner. The majority of 5`-RACE and cDNA clones isolated have contained the exon 1A sequence at their 5`-ends. This suggests that the transcript containing exon 1A is the most abundant NPY-Y1 transcript in the tissues analyzed. It remains possible that other 5`-exons represent the major transcripts in other tissues. For example, when the testis cDNA library was screened, a cDNA clone containing exon 1B was isolated, although no clones containing exon 1A or 1C were obtained.
The sequences upstream of the 5`-exons were analyzed for possible response elements for transcription factors. The transcription start sites were first determined by primer extension. Although it has previously been reported that a major transcription start site occurs 208 bp upstream of the start codon(14) , this did not correspond with any of the transcription start sites we observed using primer extension and 5`-RACE on kidney and SK-N-MC RNA. The 208-bp start site was determined by primer extension using a primer annealing to exon 2 of the gene and did not identify which 5`-exon was involved. It is possible that the transcription start site represents an additional 5`-exon sequence and/or the utilization of a different transcription start site that was undetected in this study.
None of the three promoter regions for the human NPY-Y1 gene had typical TATA or CCAAT boxes close to the transcription start sites. Many other G protein-coupled receptor genes also lack these motifs in their promoters, including the genes for the rat bradykinin B2 receptor, human adenosine A1 receptor, as well as numerous examples from the adrenergic, dopamine, and serotonin receptor families (29, 30, 31) . More than one transcription start site, as seen with exon 1C, is often a feature of these genes. The promoter preceding exon 1B has a very high G+C content that, along with the absence of TATA and CCAAT boxes, is characteristic of promoters for ``housekeeping'' genes.
Little is known
about the transcriptional regulation of the NPY-Y1 receptor gene.
Administration of glucocorticoids to rats causes an up-regulation of
NPY-Y1 receptor expression in the arcuate nucleus, although it is not
known if this is a direct effect of the glucocorticoids acting on the
NPY-Y1 receptor gene promoters(32) . However, in the human
gene, there are potential binding sites for the glucocorticoid receptor
in promoters B and C. An increase in NPY-Y1 mRNA levels in the rat
spinal cord is observed in response to peripheral
inflammation(33) . The transcription factor NF-B is an
important regulator of genes activated during the immune response, and
potential binding sites for this transcription factor exist in promoter
A of the human gene as well as in the promoter for the murine NPY-Y1
receptor gene.
The studies reported here demonstrate the existence of multiple transcripts of the human NPY-Y1 receptor mRNA, which are generated by the alternative splicing of three different 5`-exons onto a splice acceptor site within the 5`-UTR of the gene. The transcription of this receptor gene is thus under the control of at least three promoters that are activated in a tissue-specific manner and that may play an important role in the regulation of expression of the NPY-Y1 receptor and its function in a wide variety of physiological responses.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L47167[GenBank], L47168[GenBank], L47169[GenBank].