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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 15, 2001 as doi:10.1096/fj.01-0487fje. |
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Department of Pathology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA
2Correspondence: Department of Pathology, 899 Madison Ave., Room 576M, Memphis, TN 38163, USA. E-mail: aslominski{at}utmem.edu
SPECIFIC AIMS
We characterized new isoforms of human and mouse CRH-R1 and demonstrated a polymorphism of their expression.
PRINCIPAL FINDINGS
1. Alternative splicing of human CRH-R1
Four isoforms of the human CRH receptor type 1 have been described: CRH-R1
(lacking exon 6), CRH-R1ß (containing all 14 exons), CRH-R1c (lacking exons 3 and 6), and CRH-R1d (lacking exons 6 and 13). To study alternative splicing, two sets of nested primers were designed to amplify the regions of human CRH-R1 mRNA spanning exons 2 through 7 and 9 through 14 (Fig. 1
). CRH-R1 mRNA expression was detected in all human tissues and cell lines tested including pituitary, adrenal gland, skin, neonatal melanocytes and keratinocytes, immortalized HaCaT keratinocytes, squamous cell carcinoma, and melanoma cell lines. In addition to CRH-R1, c, and d already described, we identified four new isoforms: e, f, g, and h. All have exon 6 spliced out from the final transcript; in CRH-R1e, exons 3 and 4 are spliced out. CRH-R1f has a deletion of exon 12. In CRH-R1g exon 11, 27 bp of exon 10 and 28 bp of exon 12 are deleted from the mRNA transcript. CRH-R1h contains a cryptic exon (110 bp) between exons 4 and 5 (Fig. 1)
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2. Predicted protein sequences of new isoforms
CRH-R1e and CRH-R1f have frameshifts (Fig. 1)
. CRH-R1e mRNA has two potential reading frames, one of 585 bp and the second of 723 bp. The first one encodes 194 aa protein (CRH-R1e1), i.e., the first 40 aa from NH2 terminus encoded by exons 1 and 2, and a unique amino acid sequence without transmembrane binding domains. The second reading frame can potentially code a membrane-bound protein of 240 aa (CRH-R1e2) starting from the third transmembrane domain and containing the carboxyl terminus. The reading frame for CRH-R1f is 1113 bp long and encodes a receptor protein of 370 aa containing the entire CRH binding domain, the first five transmembrane domains (215 aa), and a unique carboxyl-terminal sequence. CRH-R1g has preserved the reading frame of the CRH receptor. It encodes a membrane-bound protein of 341 aa that has a deletion of 74 amino acids (transmembrane domains 5 and 6). Insertion of a 110 bp cryptic exon between exons 4 in CRH-R1h isoform would generate a truncated protein of 145 aa having only a CRH binding domain (coded by exons 1–4).
3. Differential expression pattern of CRH-R1
CRH-R1 was the most widely expressed isoform, detected in pituitary and adrenal gland, all cell lines, and most skin samples (Table 1
). CRH-R1c isoform was expressed only in basal cell carcinoma biopsy. CRH-R1d isoform was detected in the pituitary, neonatal normal keratinocytes, and five melanoma lines. CRH-R1e was detected in skin basal cell carcinoma and in four melanoma lines. CRH-R1f was detected in skin tumors and melanoma and keratinocyte lines. CRH-R1g was widely detected in pituitary, adrenals, skin, and keratinocytes and melanoma lines. The isoform CRH-R1ß was not detect in corporal skin.
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4. Environmental regulation of human CRH-R1 splicing
UV irradiation changed the spectrum of the CRH-R1 isoforms (Table 2
). In SKMEL188, UV switched expression from the CRH-R1d isoform to CRH-R1 and CRH-R1g. In HaCaT keratinocytes, first exposure to UV increased the expression of CRH-R1 without changing the isoform pattern. In C4–1 cells, UV inhibited expression of CRH-R1f and induced CRH-R1g. Since 
50% of cells exposed to UV die within 3 days after treatment, we investigated the CRH-R1 splicing pattern in cells that began rapid growth 2–3 wk after the irradiation. We found that cultured human melanoma preserved UV-induced CRH-R1 and CRH-R1g expression, but expression of the CRH-R1 isoform was increased. HaCaT keratinocytes gained CRH-R1c and g isoforms after a second UV treatment and CRH-R1e after a third UV treatment, with similar results in C4–1 cells. Thus, repeated UV treatment of the epithelial cells (HaCaT and C1–4 cells) increases expression of CRH-R1 isoforms (Table 2)
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We tested the effect of cAMP-dependent and TPA-induced pathways on CRH-R1 expression using TPA, forskolin, or a mixture of IBMX and dbcAMP (Table 2)
. In human melanoma cells, TPA shifted CRH-R1 splicing from CRH-R1d to CRH-R1 isoform. Forskolin or dbcAMP plus IBMX, like UVB, inhibited expression of CRH-R1d and stimulated expression of CRH-R1 and g isoforms. In the C4–1 cell line, these compounds had the same effects as UV. In HaCaT keratinocytes, TPA induced an insertion of 110 bp fragment between exons 4 and 5 of CRH-R1 (CRH-R1h) leading to a premature translation termination. Forskolin by itself had no effect on splicing whereas dbcAMP plus IBMX induced expression of CRH-R1e and g forms. Combinations with forskolin stimulated CRH-Rg isoform expression in most cell lines and increased CRH-R1.
5. Alternative splicing of CRH-R1 in mouse
So far, only one CRH-R1 isoform has been described in mice, an analog of human CRH-R1. We screened mouse tissue samples with a set of specific primers whose exonal location is based on rat gene structure (Table 1
; Fig. 1
). The detected isoforms have been marked in the text with the letter m in lowercase. Amplification of mouse cDNA showed mRNA for mCRH-R1 expressed in brain, pituitary, spleen, anagen IV, V, and VI skin, normal melanocytes, and Cloudman S91 melanoma cells. The isoform was absent in telogen skin. Three new isoforms were detected. One, analogous to human CRH-R1c isoform, lacks part of the CRH binding domain due to the absence of exon 3 (Fig. 1)
. This encodes a protein of 375 aa that would have decreased affinity for CRH. It is expressed only in anagen (IV-VI) skin and spleen. The other two are homologues of human CRH-R1e and CRH-R1f. Those two also have deletions of exons 3 plus 4 and exon 11, respectively, leading to frameshifts. Mouse CRH-R1e mRNA has two potential reading frames, one of 420 bp and another of 930 bp. The first one encodes a protein (mCRH-R1e1) of 139 aa that (similar to the human counterpart) contains only the first 40 aa from the NH2 terminus of the CRH-R1 without transmembrane domains. The second frame can potentially encode a membrane-bound protein of 309 aa (mCRH-R1e2) from the first transmembrane domain to the carboxyl terminus. Reading frame for mCRH-R1f (990 bp) encodes a receptor protein of 329 aa containing an entire CRH binding domain, seven transmembrane domains, and a proximal part of carboxyl terminus; the distal part of carboxyl terminus is missing due to the absence of exon 12 (Fig. 1)
. mCRH-R1e is expressed in brain, pituitary, telogen, and anagen skin; mCRH-R1f is expressed in anagen skin and the M3 subline of Cloudman S91 melanoma.
CONCLUSIONS
We have found four new types of human CRH-R1 mRNA—CRH-R1e, f, g, and h—and three new mouse isoforms homologous to human CRH-R1c, e, and f. Alternative splicing generates different mRNAs, increasing the coding capacity of genes. Approximately 33–59% of human genes have at least two variants, and 15% of the point mutations that cause diseases in humans alter the normal splicing pattern. Thus, the described spectrum of CRH-R1 isoforms in human and mouse may reflect the diverse intracellular pathways and phenotypic functions for CRH-related peptides.
Human and mouse CRH-R1e isoforms contain two reading frames, of which one encodes soluble proteins of 194 aa in humans and 139 aa in mice containing the first 40 aa of distal amino-terminal sequence with a remaining sequence different from the CRH-R1 receptor due to the frameshift. Because of the lack of exons 3, 4 and transmembrane domains, it should not act as a CRH binding protein. The second form (human: 240 aa; mouse: 309 aa) with a sequence starting from the third transmembrane domain in humans and first transmembrane domain in the mouse contains the carboxyl terminus. It will not be able to bind a ligand because of the lack of an NH2 terminus. Human CRH-R1f encodes an entire CRH binding domain and the first five transmembrane domains; therefore, it should bind CRH and fix it on the outer surface of cellular membrane. It may thus decrease local concentration of CRH or serve as a pool of bound hormone. The murine form of this receptor also encodes the entire NH2 terminus and five transmembrane domains. CRH-R1g, in which the reading frame was preserved but the protein sequence had a deletion of 74 amino acids corresponding to transmembrane domains 5 and 6, can potentially be coupled to cAMP production. Finally, CRH-R1h encodes a truncated protein having only a CRH binding domain and can potentially interfere with CRH binding or serve as an analog of CRH binding protein. The mouse tissues tested did not express CRH-R1g and h forms.
CRH-R1 is the most efficient receptor variant transducing peptide signal into cAMP-mediated pathways; other forms (ß, c, d) either have a decreased ligand binding capacity (ß, c) or are poorly coupled to cAMP production (ß, d). CRH-R1 was the most prevalent form. Its dominant role in the skin is underscored by its induction or stimulation by UV and factors raising cAMP. It also has hair cycle-dependent expression in murine skin. The second most frequent form detected in human tissues was CRH-R1g, which can potentially be coupled to the cAMP production. We suggest that the main CRH pathway in the skin involves increased production of cAMP.
The changes in receptor splicing pattern in UV irradiated skin lines support CRH action counteracting environmental stress. Thus, CRH-R1 mRNA splicing was changed from d to and g isoforms in the human melanoma cell line; CRH-R1 and g also increased in UV-treated normal (immortalized) and malignant keratinocytes. These isoforms are or can be coupled to cAMP production. The newly gained pattern of CRH-R1 splicing appears to be stable, e.g., it does not regress even after prolonged cultivation, probably promoting survival of cells damaged by UV radiation. Repeated treatments with UV increased CRH-R1 isoforms expressed in normal and malignant keratinocyte lines, resulting in populations expressing CRH-R1c, e, f, and g isoforms in addition to CRH-R1. Thus, repeated stress may favor the survival of cells with a diverse spectrum of CRH receptor isoforms, probably reflecting induced cellular heterogeneity, stabilizing phenotype, and resistance to external manipulation. Pawelek proposed that melanocyte response to solar radiation is highly regulated involving UV-stimulated expression, activation of MSH receptors, and increased ligand production. This molecular mechanism of UV action may extend to epidermal cells where UV induced CRH-R1 expression with a preference for the most efficient isoform and production of the respective CRH ligand.
Similar to UV radiation, factors raising intracellular cAMP increased CRH-R1 expression and switched the pattern to predominant expression of the and g isoforms. Thus, similar mechanisms may regulate CRH-R1 expression, being tightly linked to cAMP-activated signaling pathway(s). TPA also switched receptor splicing, but in a different pattern. We therefore suggest that cutaneous CRH-R1 gene expression can be regulated by at least two different signaling systems: one activated by UV and cAMP, and the second by TPA.
In summary, CRH-R1 is differentially spliced in a variety of human and mouse tissues. Expression of the new isoforms appears to be related to anatomic location, skin physiological and pathological status, cell type, external stress (UV), and intracellular pathway modifiers.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0487fje; to cite this article, use FASEB J. (October 15, 2001) 10.1096/fj.01-0487fje 
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