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• W2060135457 abstract "The coding region of the hamster corticotropin releasing factor receptor type 1 was sequenced. Hamster gene appeared to be similar to mouse, rat, and human sequences with 95%, 94%, and 91% homology, respectively. Protein substitutions were generally found in the corticotropin releasing factor-binding domain. Thus, this domain can be more prone to mutations leading to changes in amino acid sequence. Hamster pituitary, eye, spleen, heart, skin, and four melanoma lines differentially expressed nine corticotropin releasing factor-R1 isoforms. These included the corticotropin releasing factor-R1α and corticotropin releasing factor-R1d homologs of human isoforms as well as e, f, h, j, k, m, and n isoforms. Corticotropin releasing factor-R1e mRNA had deletion of exons 3 and 4, CRF-R1j of exon 5, CRF-R1f of exon 11, CRF-R1k of exon 10, CRF-R1m of exons 11 and 12, and CRF-R1n of exons 10, 11, and 12. Corticotropin releasing factor-R1h had an insertion of a cryptic exon between exons 4 and 5. Reading frames of isoforms e, f, j, k, m, and h contained frameshifts, expected to produce truncated proteins. Corticotropin releasing factor-R1n isoform preserved the reading frame, but the transmembrane domains 6, 7, and one-third of the fifth were deleted. The AbC1 hamster melanoma cell line changed the pattern of alternative splicing after irradiation with ultraviolet light or induction of melanogenesis; this suggests that corticotropin releasing factor receptor alternative splicing may be regulated by common stressors, through modifications of activity and/or availability of splicing factors. The coding region of the hamster corticotropin releasing factor receptor type 1 was sequenced. Hamster gene appeared to be similar to mouse, rat, and human sequences with 95%, 94%, and 91% homology, respectively. Protein substitutions were generally found in the corticotropin releasing factor-binding domain. Thus, this domain can be more prone to mutations leading to changes in amino acid sequence. Hamster pituitary, eye, spleen, heart, skin, and four melanoma lines differentially expressed nine corticotropin releasing factor-R1 isoforms. These included the corticotropin releasing factor-R1α and corticotropin releasing factor-R1d homologs of human isoforms as well as e, f, h, j, k, m, and n isoforms. Corticotropin releasing factor-R1e mRNA had deletion of exons 3 and 4, CRF-R1j of exon 5, CRF-R1f of exon 11, CRF-R1k of exon 10, CRF-R1m of exons 11 and 12, and CRF-R1n of exons 10, 11, and 12. Corticotropin releasing factor-R1h had an insertion of a cryptic exon between exons 4 and 5. Reading frames of isoforms e, f, j, k, m, and h contained frameshifts, expected to produce truncated proteins. Corticotropin releasing factor-R1n isoform preserved the reading frame, but the transmembrane domains 6, 7, and one-third of the fifth were deleted. The AbC1 hamster melanoma cell line changed the pattern of alternative splicing after irradiation with ultraviolet light or induction of melanogenesis; this suggests that corticotropin releasing factor receptor alternative splicing may be regulated by common stressors, through modifications of activity and/or availability of splicing factors. Corticotropin releasing factor (CRF) and related peptides coordinate the complex array of behavioral, autonomic, and endocrine responses to stress at the central level. These peptides are present in peripheral organs, including skin where they regulate local homeostasis acting as potent immunomodulators, growth factors, and regulators of vascular function (Chrousos, 1995Chrousos G.P. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation.N Engl J Med. 1995; 332: 1351-1362Crossref PubMed Scopus (2217) Google Scholar;Slominski and Wortsman, 2000Slominski A. Wortsman J. Neuroendocrinology of the skin.Endocr Rev. 2000; 21: 457-487Crossref PubMed Scopus (650) Google Scholar;Slominski et al., 2000bSlominski A. Wortsman J. Luger T. Paus R. Salomon S. Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress.Physiol Rev. 2000; 80: 979-1020Crossref PubMed Scopus (634) Google Scholar,Slominski et al., 2001Slominski A. Wortsman J. Pisarchik A. Zbytek B. Linton E.A. Mazurkiewicz J. Wei E.T. Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors.FASEB J. 2001; 15: 1678-1693Crossref PubMed Scopus (247) Google Scholar). In addition, CRF, and urocortin participate in the normal progression of pregnancy (Linton et al., 2001Linton E.A. Woodman J.R. Asboth G. Glynn B.P. Plested C.P. Bernal A.L. Corticotrophin releasing hormone: its potential for a role in human myometrium.Exp Physiol. 2001; 86: 273-281Crossref PubMed Scopus (31) Google Scholar). These diverse phenotypic effects of CRF peptides are mediated through interaction with G protein coupled membrane bound CRF receptors (Perrin and Vale, 1999Perrin M.H. Vale W.W. Corticotropin releasing factor receptors and their ligand family.Ann NY Acad Sci. 1999; 885: 312-328Crossref PubMed Scopus (392) Google Scholar). So far three receptor subtypes have been characterized with CRF-R1 and CRF-R2 detected in mammals and Xenopus (Chang et al., 1993Chang C.P. Pearse R.V. O'Connell S. Rosenfeld M.G. Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain.Neuron. 1993; 11: 1187-1195Abstract Full Text PDF PubMed Scopus (510) Google Scholar;Chen et al., 1993Chen R. Lewis K.A. Perrin M.H. Vale W.W. Expression cloning of a human corticotropin-releasing-factor receptor.Proc Natl Acad Sci USA. 1993; 90: 8967-8971Crossref PubMed Scopus (901) Google Scholar;Lovenberg et al., 1995Lovenberg T.W. Liaw C.W. Grigoriadis D.M. Clevenger W. Chalmers D.T. De Souza E.B. Oltersdorf T. Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain.Proc Natl Acad Sci USA. 1995; 92: 836-840Crossref PubMed Scopus (831) Google Scholar;Perrin et al., 1995Perrin M. Donaldson C. Chen R. et al.Identification of a second corticotropin-releasing factor receptor gene and characterization of a cDNA expressed in heart.Proc Natl Acad Sci USA. 1995; 92: 2963-2973Crossref Scopus (492) Google Scholar;Liaw et al., 1996Liaw C.W. Lovenberg T.W. Barry G. Oltesdorf T. Grigoriadis D.E. De Souza E.B. Cloning and characterization of the human corticotropin-releasing factor-2 receptor complementary deoxyribonucleic acid.Endocrinology. 1996; 137: 72-77Crossref PubMed Google Scholar;Vita et al., 1993Vita N. Laurent P. Lefort S. et al.Primary structure and functional expression of mouse pituitary and human brain corticotrophin releasing factor receptors.FEBS Lett. 1993; 335: 1-5Abstract Full Text PDF PubMed Scopus (298) Google Scholar;Stenzel et al., 1995Stenzel P. Kesterson R. Yeung W. Cone R.D. Identification of a novel murine receptor for corticotropin-releasing hormone expressed in the heart.Mol Endocrinol. 1995; 9: 637-645Crossref PubMed Google Scholar;Dautzenberg et al., 1997Dautzenberg F.M. Dietrich K. Palchaudhuri M.R. Spiess J. Identification of two corticotropin-releasing factor receptors from Xenopus laevis with high ligand selectivity: unusual pharmacology of the type 1 receptor.Neurochemistry. 1997; 69: 1640-1649Crossref PubMed Scopus (96) Google Scholar), whereas CRF-R3 is detected only in catfish (Arai et al., 2001Arai M. Assil I.Q. Abou-Samra A.B. Characterization of three corticotropin-releasing factor receptors in catfish: a novel third receptor is predominantly expressed in pituitary and urophysis.Endocrinology. 2001; 142: 446-454Crossref PubMed Scopus (137) Google Scholar). The CRF-R1 was cloned in humans, rat, mouse, sheep, tree shrew, chicken, Xenopus, and catfish (Chang et al., 1993Chang C.P. Pearse R.V. O'Connell S. Rosenfeld M.G. Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain.Neuron. 1993; 11: 1187-1195Abstract Full Text PDF PubMed Scopus (510) Google Scholar;Chen et al., 1993Chen R. Lewis K.A. Perrin M.H. Vale W.W. Expression cloning of a human corticotropin-releasing-factor receptor.Proc Natl Acad Sci USA. 1993; 90: 8967-8971Crossref PubMed Scopus (901) Google Scholar;Vita et al., 1993Vita N. Laurent P. Lefort S. et al.Primary structure and functional expression of mouse pituitary and human brain corticotrophin releasing factor receptors.FEBS Lett. 1993; 335: 1-5Abstract Full Text PDF PubMed Scopus (298) Google Scholar;Yu et al., 1996Yu J. Xie L.Y. Abou-Samra A.B. Molecular cloning of a type A chicken corticotropin-releasing factor receptor with high affinity for urotensin I.Endocrinology. 1996; 137: 192-197Crossref PubMed Scopus (68) Google Scholar;Dautzenberg et al., 1997Dautzenberg F.M. Dietrich K. Palchaudhuri M.R. Spiess J. Identification of two corticotropin-releasing factor receptors from Xenopus laevis with high ligand selectivity: unusual pharmacology of the type 1 receptor.Neurochemistry. 1997; 69: 1640-1649Crossref PubMed Scopus (96) Google Scholar;Myers et al., 1998Myers D.A. Trinh J.V. Myers T.R. Structure and function of the ovine type 1 corticotropin releasing factor receptor (CRF1) and a carboxyl-terminal variant.Mol Cell Endocrinol. 1998; 144: 21-35Crossref PubMed Scopus (35) Google Scholar;Palchaudhuri et al., 1998Palchaudhuri M.R. Wille S. Mevenkamp G. Spiess J. Fuchs E. Dautzenberg F.M. Corticotropin-releasing factor receptor type 1 from Tupaia belangeri-cloning, functional expression and tissue distribution.Eur J Biochem. 1998; 258: 78-84Crossref PubMed Scopus (61) Google Scholar;Arai et al., 2001Arai M. Assil I.Q. Abou-Samra A.B. Characterization of three corticotropin-releasing factor receptors in catfish: a novel third receptor is predominantly expressed in pituitary and urophysis.Endocrinology. 2001; 142: 446-454Crossref PubMed Scopus (137) Google Scholar). The genes for human (GenBank accession nos AF039510–AF3523) and rat CRF-R1 (GenBank accession nos U53486–U53498) contain 14 and 13 exons, respectively (Sakai et al., 1998Sakai K. Yamada M. Horiba N. Wakui M. Demura H. Suda T. The genomic organization of the human corticotropin-releasing factor type-1 receptor.Gene. 1998; 219: 125-130Crossref PubMed Scopus (34) Google Scholar;Tsai-Morris et al., 1996Tsai-Morris C.H. Buczko E. Geng Y. Gamboa-Pinto A. Dufau M.L. The genomic structure of the rat corticotropin releasing factor receptor. A member of the class II G protein-coupled receptors.J Biol Chem. 1996; 271: 14519-14525Crossref PubMed Scopus (56) Google Scholar), and produce several alternatively spliced isoforms. In humans four CRF-R1 isoforms have been described: CRF-R1α (lacking exon 6), CRF-R13β (containing all 14 exons), CRF-R1c (lacking exons 3 and 6), and CRF-R1d (lacking exons 6 and 13) (Chen et al., 1993Chen R. Lewis K.A. Perrin M.H. Vale W.W. Expression cloning of a human corticotropin-releasing-factor receptor.Proc Natl Acad Sci USA. 1993; 90: 8967-8971Crossref PubMed Scopus (901) Google Scholar;Ross et al., 1994Ross P.S. Kostas C.M. Ramabhadran T.V. A variant of the human corticotropin-releasing factor (CRF) receptor: cloning, expression and pharmacology.Biochem Biophys Res Commun. 1994; 205: 1836-1842Crossref PubMed Scopus (77) Google Scholar;Grammatopoulos et al., 1999Grammatopoulos D.K. Dai Y. Randeva H.S. Levine M. Karteris E. Easton A. Hillhouse E.W. A novel spliced variant of the type 1 corticotropin-releasing hormone receptor with a deletion in the seventh transmembrane domain present in the human pregnant term myometrium and fetal membranes.Mol Endocrinol. 1999; 13 (2122): 2189Crossref PubMed Scopus (116) Google Scholar). In rats, however, there are three CRF-R1 isoforms: A spanning 1–13 exons, B with deletion of exon 3, and C with deletion of exons 7, 11, and 12 (Tsai-Morris et al., 1996Tsai-Morris C.H. Buczko E. Geng Y. Gamboa-Pinto A. Dufau M.L. The genomic structure of the rat corticotropin releasing factor receptor. A member of the class II G protein-coupled receptors.J Biol Chem. 1996; 271: 14519-14525Crossref PubMed Scopus (56) Google Scholar). In the mouse, only one isoform, an equivalent to human CRF-R1α has been characterized (Vita et al., 1993Vita N. Laurent P. Lefort S. et al.Primary structure and functional expression of mouse pituitary and human brain corticotrophin releasing factor receptors.FEBS Lett. 1993; 335: 1-5Abstract Full Text PDF PubMed Scopus (298) Google Scholar). Recently, four additional CRF-R1 mRNA types for humans were described: e, f, g, and h (corresponding gene accession nos are AF369651, AF369652, AF369653, and AF374231), and three for mouse that are homologous to human CRF-R1c, e, and f isoforms (corresponding gene accession nos are AF369654, AF369655, AF369656) (Pisarchik and Slominski, 2001Pisarchik A. Slominski A. Alternative splicing of CRH-R1 receptors in human and mouse skin: identification of new variants and their differential expression.in: FASEB J. 15. 2001: 2754-2756https://doi.org/10.1096/fj.01-0487fjeCrossref Scopus (159) Google Scholar). It has been found that additional interspecies polymorphism of CRF-R1 isoform expression is related to anatomic location, skin physiologic or pathologic status, specific cell type, and presence or absence of a common skin stressor [ultraviolet (UV) radiation] (Pisarchik and Slominski, 2001Pisarchik A. Slominski A. Alternative splicing of CRH-R1 receptors in human and mouse skin: identification of new variants and their differential expression.in: FASEB J. 15. 2001: 2754-2756https://doi.org/10.1096/fj.01-0487fjeCrossref Scopus (159) Google Scholar). To define further the role of CRF-R1 in skin the complementary cDNA in Syrian golden hamsters (Mesocricetus aureatus) was cloned. These rodents are routinely used in our laboratory to study the behavior of transplantable melanomas and their interaction with the host organism (Bomirski et al., 1988Bomirski A. Slominski A. Bigda J. The natural history of a family of transplantable melanomas in hamsters.Cancer Metastasis Rev. 1988; 7: 95-118Crossref PubMed Scopus (89) Google Scholar;Slominski and Paus, 1993Slominski A. Paus R. Bomirski melanomas. A versatile and powerful model for pigment cell and melanoma research.Int J Oncol. 1993; 2: 221-228PubMed Google Scholar). One of the models, the Bomirski AbC1 hamster melanoma cell line is a powerful experimental system for the study of environmental regulation of cell differentiation and melanogenesis (Slominski et al., 1988Slominski A. Moellmann G. Kuklinska E. Bomirski A. PawelekJ. Positive regulation of melanin pigmentation by two key substrates of the melanogenic pathway, 1-tyrosine and 1-dopa.J Cell Sci. 1988; 89: 287-296PubMed Google Scholar,Slominski et al., 1989Slominski A. Moellmann G. Kuklinska E. MSH inhibits growth in a line of amelanotic hamster melanoma cells and induces increases in cyclic AMP levels and tyrosinase activity without inducing melanogenesis.J Cell Sci. 1989; 92: 551-559PubMed Google Scholar). This study reports the full cDNA sequence of the hamster CRF-R1 receptor and characterized alternative splicing patterns in skin. Hamster eyes, pituitary, heart, spleen, skin as well as four lines of hamster melanoma were used for the experiments. Syrian hamsters (males 3 mo old) were purchased from Taconic (New York) and housed in community cages at the animal facilities of the Albany Medical College (AMC), Albany NY. The animals were killed under pentobarbital anesthesia and selected organs as well as back skin were collected following protocols routinely used in our laboratory (Slominski et al., 1996Slominski A. Baker J. Rosano T. Guisti L.W. Ermak G. Grande M. Gaudet S.J. Metabolism of serotonin to N-acetylserotonin, melatonin, and 5-methoxytryptamine in hamster skin culture.J Biol Chem. 1996; 271: 12281-12286Crossref PubMed Scopus (101) Google Scholar). Tissue specimens were frozen rapidly in liquid nitrogen. Hamster Bomirski Ma melanotic, MI hypomelanotic and Ab amelanotic melanomas were propagated in male Syrian hamsters by subcutaneous inoculation of tissue suspension as described previously (Bomirski et al., 1988Bomirski A. Slominski A. Bigda J. The natural history of a family of transplantable melanomas in hamsters.Cancer Metastasis Rev. 1988; 7: 95-118Crossref PubMed Scopus (89) Google Scholar). After killing the animals tumor tissue was freed from connective and necrotic tissues and frozen rapidly in liquid nitrogen. Hamster tissues as well as melanoma transplants were stored at -80°C until further analysis. The experimental protocol was originally approved by the Institutional Animal Care and Use Committee at AMC, and a similar protocol for mice was approved at the University of Tennessee Health Science Center. Bomirski AbC-1 hamster melanoma cells were grown in Ham's F10 medium as described previously; the media were supplemented with 10% fetal bovine serum and antibiotics (Gibco BRL, Gaithersburg, MD) (Slominski et al., 1988Slominski A. Moellmann G. Kuklinska E. Bomirski A. PawelekJ. Positive regulation of melanin pigmentation by two key substrates of the melanogenic pathway, 1-tyrosine and 1-dopa.J Cell Sci. 1988; 89: 287-296PubMed Google Scholar). To induce melanogenesis the cells were cultured for 3 d in Dulbecco's minimal Eagle's medium plus 10% fetal bovine serum. Melanoma cells were detached with Tyrode's solution containing 1 mM ethylenediamine tetraacetic acid after prior washing with phosphate-buffered saline (Slominski et al., 1988Slominski A. Moellmann G. Kuklinska E. Bomirski A. PawelekJ. Positive regulation of melanin pigmentation by two key substrates of the melanogenic pathway, 1-tyrosine and 1-dopa.J Cell Sci. 1988; 89: 287-296PubMed Google Scholar). The cells were centrifuged at 4°C, washed with cold phosphate-buffered saline and cell pellets were used for RNA isolation. UV irradiation of AbC1 cells was performed as described previously for human cell lines (Pisarchik and Slominski, 2001Pisarchik A. Slominski A. Alternative splicing of CRH-R1 receptors in human and mouse skin: identification of new variants and their differential expression.in: FASEB J. 15. 2001: 2754-2756https://doi.org/10.1096/fj.01-0487fjeCrossref Scopus (159) Google Scholar). Briefly, cells were cultured in 9 cm Petri dishes at concentration 106 cells per dish and before irradiation, medium was aspirated and substituted by 10 ml of phosphate-buffered saline. The dishes were placed on the UV transilluminator 2000 (Bio-Rad, Hercules, CA) and incubated for 30 s corresponding to UVB doses of 50 mJ per cm2. Time of exposure and corresponding doses as well as the spectrum of UV have been established previously (Pisarchik and Slominski, 2001Pisarchik A. Slominski A. Alternative splicing of CRH-R1 receptors in human and mouse skin: identification of new variants and their differential expression.in: FASEB J. 15. 2001: 2754-2756https://doi.org/10.1096/fj.01-0487fjeCrossref Scopus (159) Google Scholar). After irradiation phosphate-buffered saline was substituted by standard culture medium, cells were incubated for 24 h, and then detached, collected, and processed for RNA isolation. Total RNA was extracted using Trizol isolation kit (Gibco-BRL). The synthesis of first-strand cDNA was performed using the Superscript preamplification system (Gibco-BRL). A 5 g of total RNA per reaction was reverse transcribed using oligo(dT) as the primer. The areas spanning exons 2–6 and exons 8–13 of CRF-R1 mRNA (Figure 1) are more prone to undergo alternative splicing (Chen et al., 1993Chen R. Lewis K.A. Perrin M.H. Vale W.W. Expression cloning of a human corticotropin-releasing-factor receptor.Proc Natl Acad Sci USA. 1993; 90: 8967-8971Crossref PubMed Scopus (901) Google Scholar;Ross et al., 1994Ross P.S. Kostas C.M. Ramabhadran T.V. A variant of the human corticotropin-releasing factor (CRF) receptor: cloning, expression and pharmacology.Biochem Biophys Res Commun. 1994; 205: 1836-1842Crossref PubMed Scopus (77) Google Scholar;Tsai-Morris et al., 1996Tsai-Morris C.H. Buczko E. Geng Y. Gamboa-Pinto A. Dufau M.L. The genomic structure of the rat corticotropin releasing factor receptor. A member of the class II G protein-coupled receptors.J Biol Chem. 1996; 271: 14519-14525Crossref PubMed Scopus (56) Google Scholar;Grammatopoulos et al., 1999Grammatopoulos D.K. Dai Y. Randeva H.S. Levine M. Karteris E. Easton A. Hillhouse E.W. A novel spliced variant of the type 1 corticotropin-releasing hormone receptor with a deletion in the seventh transmembrane domain present in the human pregnant term myometrium and fetal membranes.Mol Endocrinol. 1999; 13 (2122): 2189Crossref PubMed Scopus (116) Google Scholar). These two mRNA regions were amplified by nested reverse transcription–PCR to characterize hamster CRF-R1 isoforms. The first round of amplification was done using 2 μl of cDNA. Aliquot of PCR mixture from the first round of amplification was transferred to a new tube and a second round of PCR was conducted. For the region from exon 2 though exon 6 primers P156 and P157 were used in the first round of PCR and primers P158 and P159 for the second one. Exons 8–13 were amplified by primers P160 and P161 in the first round and by primers P162 and P163 in the second one. The reaction mixture (25 μl) contained 2.5 mM MgCl2, 2.5 mM of each deoxyribonucleoside triphosphate, 0.4 M of each primer, 75 mM Tris–HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween 20, and 0.25 units of Taq polymerase. The mixture was heated to 94°C for 2.5 min and then amplified for 35 cycles: 94°C for 30 s (denaturation), 65°C for 45 s (annealing), and 72°C for 1 min (extension). Amplification products were separated by agarose electrophoresis and visualized by ethidium bromide staining according to standard protocol used in our laboratory (Slominski et al., 2000aSlominski A. Roloff B. Curry J. Dahiya M. Szczesniewski A. Wortsman J. The skin produces urocortin.J Clin Endocrinol Metab. 2000; 85: 815-823PubMed Google Scholar). All bands were excised from the gel, cloned in pGEM-Teasy vector (Promega, Madison, WI), and sequenced. All samples were analyzed and standardized by the amplification of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase as described previously byRobbins and McKinney, 1992Robbins M. McKinney M. Transcriptional regulation of neuromodulin (GAP-43) in mouse neuroblastoma clone N1E-115 as evaluated by the RT/PCR method.Brain Res Mol Brain Res. 1992; 13: 83-92Crossref PubMed Scopus (54) Google Scholar). The sequences of primers were as follows P156: 5′-TCCGGCTCGTGAAGGCCCTTC-3′ (sense, exon 2) P157: 5′-GCTCAGGGTGAGCTGGACCAC-3′ (anti-sense, exon 6) P158: 5′-TGTCCCTGGCCAGCAATGTCTC-3′ (sense, exon 2) P159: 5′-AGTGGATGATGTTCCTCAGGCAC-3′ (anti-sense, exon 6) P160: 5′-CCATTGGGAAACTTTACTACGAC-3′ (sense, exon 8) P161: 5′-CTTGATGCTGTGGAAGCTGACTC-3′ (anti-sense, exon 13) P162: 5′-AAAAGTGCTGGTTTGGCAAACGTC-3′ (sense, exon 8) P163: 5′-CTTCCGGATGGCAGAGCGGAC-3′ (anti-sense, exon 13) P170: 5′-ACAGAGCGAGCCCGAGGATG-3′ (sense) P171: 5′-TCGGGTCTTGGGGGCTGCTC-3′ (anti-sense) P179: 5′-ATGGGACAGCGCCCGCAGCTC-3′ (sense). A full-length coding region of hamster CRF receptor mRNA was obtained by reverse transcription–PCR of hamster pituitary cDNA by primers that were designed from the published sequence of mouse gene (Vita et al., 1993Vita N. Laurent P. Lefort S. et al.Primary structure and functional expression of mouse pituitary and human brain corticotrophin releasing factor receptors.FEBS Lett. 1993; 335: 1-5Abstract Full Text PDF PubMed Scopus (298) Google Scholar). The structure of rat CRF-R1 gene is different from the human one. Human gene has 14 exons (Sakai et al., 1998Sakai K. Yamada M. Horiba N. Wakui M. Demura H. Suda T. The genomic organization of the human corticotropin-releasing factor type-1 receptor.Gene. 1998; 219: 125-130Crossref PubMed Scopus (34) Google Scholar) and rat only 13 (Tsai-Morris et al., 1996Tsai-Morris C.H. Buczko E. Geng Y. Gamboa-Pinto A. Dufau M.L. The genomic structure of the rat corticotropin releasing factor receptor. A member of the class II G protein-coupled receptors.J Biol Chem. 1996; 271: 14519-14525Crossref PubMed Scopus (56) Google Scholar). The composition of exons in the hamster gene was presumed to be similar to rat as hamster is supposed to be a closer relative to this species. Sequencing strategy is showed in the Figure 1. First, the coding sequence spanning exons 3–12 was amplified by nested PCR; primers P156 and P161 were used in the first round of amplification and primers P158 and 163 in the second round. A band spanning exons 1–5 was produced by nested PCR with primers P170 and P157 in the first round of amplification and P179 and P159 in the second. Primers P162 and P171 amplified the fragment containing exons 9–13 (Figure 1). All these bands were cloned in pGEM-Teasy vector (Promega) and sequenced. The sequences were aligned producing the full-length coding region of hamster CRF-R1 gene. The identified PCR products were excised from the agarose gel and purified by GFX PCR DNA and gel band purification kit (Amersham-Pharmacia-Biotech, Piscataway, NJ). PCR fragments were cloned in pGEM-Teasy vector according to the manufacturer's protocol (Promega). Plasmid DNA was purified by Plasmid Mini Kit (Qiagen, Valencia, CA). Sequencing was performed in the Molecular Resource Center at the University of Tennessee HSC (Memphis) using an Applied Biosystems 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) and BigDye Terminator Kit. Rat CRF-R1 gene was characterized previously and was found to have 13 exons in contrast to 14 exons in humans (Tsai-Morris et al., 1996Tsai-Morris C.H. Buczko E. Geng Y. Gamboa-Pinto A. Dufau M.L. The genomic structure of the rat corticotropin releasing factor receptor. A member of the class II G protein-coupled receptors.J Biol Chem. 1996; 271: 14519-14525Crossref PubMed Scopus (56) Google Scholar;Sakai et al., 1998Sakai K. Yamada M. Horiba N. Wakui M. Demura H. Suda T. The genomic organization of the human corticotropin-releasing factor type-1 receptor.Gene. 1998; 219: 125-130Crossref PubMed Scopus (34) Google Scholar). We have presumed that the organization of exons in hamster CRF-R1 gene would be similar to rat and counted exons accordingly. As a first step of hamster CRF-R1 cloning, we amplified hamster skin with the primers that were designed previously for the mouse (Pisarchik and Slominski, 2001Pisarchik A. Slominski A. Alternative splicing of CRH-R1 receptors in human and mouse skin: identification of new variants and their differential expression.in: FASEB J. 15. 2001: 2754-2756https://doi.org/10.1096/fj.01-0487fjeCrossref Scopus (159) Google Scholar). The central region of hamster gene, containing exons 3–12, was amplified and sequenced (Figure 1). Several approaches were implemented to amplify exons 1, 2, and 13 of hamster CRF receptor gene. 5′- and 3′-RACE did not produce clear bands. Thus, as an alternative we designed primers to mouse CRF-R1 5′- and 3′ uncoding regions as well as a primer starting from an initiation codon (primer P179) (Figure 1). Nested PCR produced two bands containing exons 1–5 and 9–13 (Figure 1). All the bands were sequenced and aligned with the central fragment, and the full-length coding region of the hamster gene was received. Hamster CRF-R1 coding sequence appeared to be very similar to already cloned sequences as compared with several CRF-R1 genes available in the GenBank database using DNAMLK program from the PHYLIP package (Felsenstein, 1993Felsenstein J. PHYLIP, Version 3.5c., Distributed by the author. Department of Genetics, University of Washington, Seattle1993Google Scholar). In agreement with our assumption, mouse and rat were the closest relatives of hamster among the tested species (Figure 2). Thus, the hamster sequence has 95%, 94%, and 91% homologies with mouse, rat, and human sequences, respectively. Comparison of deduced amino acid sequence of hamster CRF-R1 with the mouse, rat, and human receptors showed an even higher degree of homology (98%, 97%, and 97%, respectively). Over 80% of nucleotide substitutions did not affect amino acid sequence implying the existence of some stabilizing selection. Nucleotide substitutions were evenly spread across the mRNA sequence, but 11 of 18 changeable amino acids (61%) were situated in the extracellular domain, which is involved in CRF binding (Figure 3) (Perrin et al., 1998Perrin M.H. Sutton S. Bain D. Berggren W.T. Vale W.W. The first extracellular domain of corticotropin releasing factor-R1 contains major binding determinants for urocortin and astressin.Endocrinology. 1998; 139: 566-570Crossref PubMed Scopus (95) Google Scholar). Thus, this domain might be more susceptible to mutations leading to changes in amino acid sequence.Figure 3Alignment of mouse CRF-R1α (accession no. NM_007762), rat CRF-R1A (accession no. L25438), human CRF-R1α (accession no. L23332), and a predicted amino acid sequence of hamster CRF-R1α (GenBank accession no. AY034599). Conserved amino acids are shadowed. Arrows indicate the positions of introns. The putative transmembrane domains are indicated by rows of # symbols below the appropriate amino acids. The numbers in the right-hand column refer to the amino acid number.View Large Image Figure ViewerDownload (PPT) Here we offer an explanation of the higher speed of evolutionary changes in the CRF-binding domain of CRF-R1. On the one hand, the substituted amino acids might be not critical for the CRF binding. Thus, it was shown that N-terminal domain contain two regions crucial for the binding of CRF receptor agonists and antagonists. One of them maps to amino acids 43–50 and the second one to amino acids 76–84 of CRF-R1 (Wille et al., 1999Wille S. Sydow S. Palchaudhuri M.R. Spiess J. Dautzenberg F.M. Identification of amino acids in the N-terminal domain of corticotropin-releasing fac" @default.
• W2060135457 created "2016-06-24" @default.
• W2060135457 creator A5003515174 @default.
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• W2060135457 title "Corticotropin Releasing Factor Receptor Type 1: Molecular Cloning and Investigation of Alternative Splicing in the Hamster Skin" @default.
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