- Email: [email protected]
Growth Hormone Gene Family
Biochemical and Biophysical Research Communications 262, 575–578 (1999) Article ID bbrc.1999.1260, available online at http://www.idealibrary.com on
Identification of Four New Members of the Rat Prolactin/Growth Hormone Gene Family Kenichi Ishibashi 1 and Masashi Imai Department of Pharmacology, Jichi Medical School, Tochigi 329-0498, Japan
Received July 14, 1999
The rodent prolactin (PRL)/growth hormone (GH) gene family currently consists of at least 14 distinct genes that are expressed mainly in pituitary, uterus, and/or placenta. We report here the identification of novel four members from rat with significant homology to PRL. The encoding proteins are not homologs of other known members of this hormone family. The four new cDNAs were assigned to PRL family based on sequence homology and were referred to as PRL-like protein-I (PLP-I), PLP-J, PLP-K, and PLP-L, following the current naming order of rodent PLP family, where PLP-H is the most recent gene. They encode amino acids with 211–228 amino acids, and 34 –38% identity with PRL. All have one or two N-linked glycosylation sites. Among the examined rat tissues by Northern blot analysis, only PLP-I was expressed in testis. Our results indicate that the rodent PRL/GH gene family is large with at least 18 distinct genes. © 1999 Academic Press
The pituitary hormones, growth hormone (GH) and prolactin (PRL), and placental lactogen (PL) of placental origin are structurally related and grouped together in the PRL/GH family (1). This family has recently been expanded due to the discovery of rodent genes. These proteins are structurally more similar to PRL (30 – 40%) than to GH (15–21%), suggesting that they may be derived from a PRL gene. Some are named PRL-like peptide (PLP). These rodent proteins are currently made of 18 different members (2). However, some seem to be derived from same gene through alternative splicing or minor strain differences. PLP-C and PLP-Cv are derived from same gene (3). PL-Iv and PL-Imix come from PL-I gene (4). PLP-E and PLP-G are virtually identical (5, 6). Therefore, currently the PRL/GH family consists of 14 distinct genes. These genes are GH, PRL, PLP-A, PLP-B, PLP-C, PLP-D, PLP-E, PLP-F (5) PLP-H (7), PL-I, PL-II, d/tPRL (decidual PRL-related protein), PLF (proliferin) (8), and 1
To whom correspondence and reprint requests should be addressed. Fax: 181-285-44-5541. E-mail: [email protected].
PLF-RP (proliferin-related peptide). Within the PLP subfamily, PLP-C, PLP-D, d/tPRL, and PLP-H are highly homologous. PLP-H is the most recently identified member (7). Recently, a novel member of this family has been discovered in fish pituitary and named somatolactin (SL) (9, 10). SL is similarly related to GH and PRL. The function of SL is not well understood, although the roles in sexual maturation stress responses and calcium and acid base regulation have been suggested. SL has a stimulatory effect on the renal net phosphate reabsorption in renal tubule primary culture (11). Currently, SL is only identified in teleost fishes. SL may be present in all vertebrate species. Moreover, the expression of SL is not limited to pituitary gland. It is also expressed in brain and widely distributed in most brain lesion (12). The structural similarity of SL to the PRL/GH family and the wide distribution in the fish brain prompted us to search for its mammalian homologs in the expressed sequence tag (EST) banks where many clones from mammalian brain cDNAs are deposited. However, we could not identify any mammalian SL homologs. Instead, we identified four more new members of this gene family. Interestingly, one of them was expressed in testis. MATERIALS AND METHODS To identify the new members of PRL/GH family, queries were made in the public dbEST database (maintained at NCBI/NIH) with the coding region of the rat PRL/GH sequences using the BLAST program. Several EST sequences were identified which have amino acid sequences related to PRL that had not been previously reported. Inclusion in the PRL family is dependent upon the positioning of four core cysteine residues and some other key amino acids. Four distinct closely related novel ESTs homologs to PRL were identified. These clones were obtained from Research Genetics (Huntsville, AL), Genome Systems (St. Louis, MO), and American Type Culture Collection (Manassas, VA). These clones were sequenced fully on both strands using fluorescent dye terminator chemistry (Applied Biosystems). Analyses of the nucleotide and amino acid sequences were performed using DNAsis v3.5 (Hitachi Software Engineering Co., Tokyo). Rat multiple tissue Northern blots were purchased from Clontech Laboratories (Palo Alto, CA) which had 2 mg polyA(1) RNA at each
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0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Vol. 262, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Multiple alignments of rat PRL (Prolactin), GH (Growth Hormone), and four new members of PLPs (prolactin-like proteins). The six cysteine residues that form a large disulfide loop and small loops near the amino- and carboxy-termini are indicated by V and X, respectively. The junctions between signal peptides and mature peptides are indicated by Y for PRL and by inverted v for GH.
lane. The membranes were hybridized in ExpressHyb hybridization solution (Clontech) for 3 h at 68°C with probes, randomly 32P-labeled full inserts of each clone containing its open reading frame. The membranes were washed in a final buffer of 0.13 SSC and 0.1% SDS, then exposed to X-ray film at 280°C between two intensifying screens. The blots were reprobed after deprobing in boiled 0.1% SDS solution for 15 min.
RESULTS AND DISCUSSION We identified four new members of PRL family proteins, which are not the currently known members of this family. They have relatively low homology with PRL up to 38%. The most recent new members of this family are PLP-H (7). Following this naming order, we tentatively named these proteins PLP-I, PLP-J, PLP-K, and PLP-L and deposited in GenBank under the accession numbers AB019791, AB019945, AB022882, and AB002283, respectively. The derived amino acid sequences were aligned in Fig. 1. PLP-I was derived from rat spleen cDNA(AA851934) of 1035 nucleotides encoding 220 amino acid residues with a 59-UTR of 241 bp, 39-UTR of134 bp, and polyA tail. A signal peptide seemed to be absent at N-terminus. The open reading frame is 34% identical
with PRL. The 13 cysteine residues were present in PLP-I and four of them can be aligned at the same position as the cysteines in PRL of other vertebrates (indicated by V and X in Fig. 1). These four cysteines will form a large disulfide loop and a small loop near the carboxy-terminus. These are one of the hallmarks of proteins in the PRL/GH family (13), while PRL has an additional small amino-terminal disulfide loop (indicated by X in Fig. 1). The presence of six cysteines in the first 35 amino acid residues of N-terminus is noteworthy. Some of them will form a small N-terminal disulfide loop after the cleavage of a putative signal peptide. PLP-I has a putative N-linked glycosylation site (residues 76-79, NSST). PLP-J was derived from rat placenta cDNA (AA875114) of 810 nucleotides encoding 211 amino acid residues with a 59-UTR of 49 bp, 39-UTR of 128 bp, and polyA tail. The open reading frame is 35% identical with PRL. The first 29 amino acid residues may be a signal peptide (boundary was indicated by Y in Fig. 1) and the mature peptide (182 amino acids) has five cysteines. Two of them will form a large disulfide loop. However, the other cysteines are not conserved. These cysteines may form covalent intramolecular interactions that are not typically found in this family. Therefore, PLP-J is unique in that it lacks both N-terminal and C-terminal small disulfide loops, which is unusual in PRL/GH family. Based on this unique secondary structure, the receptor for PLP-J may be different from that of PRL. PLP-J has two putative N-linked glycosylation sites (residues 77– 80, NNTC and residues 173–176, NKSI). PLP-K was derived from rat placenta cDNA (AI059879) of 842 nucleotides encoding 228 amino acid residues with a 59-UTR of 44 bp, 39-UTR of113 bp, and polyA tail. The open reading frame is 38% identical with PRL. The first 31 amino acid residues may be a signal peptide and the mature peptide (197 amino acids) has five cysteines. Two of them will form a large disulfide loop. As both N- and C-termini have unpaired cysteines, two small disulfide loops at both ends may not be formed. Therefore, the secondary structure of PLP-K may be similar to that of PLP-J. PLP-K has a putative N-linked glycosylation site (residues 173–176, NDTH). PLP-L was derived from rat placenta cDNA (AI028934) of 844 nucleotides encoding 228 amino acid residues with a 59-UTR of 50 bp, 39-UTR of109 bp, and polyA tail. The open reading frame is 37% identical with PRL. The first 27 amino acid residues may be a signal peptide and the mature peptide (201 amino acids) has five cysteines. Four of them will form a large disulfide loop and an N-terminal small loop. However, the C-terminal disulfide small loop, which is conserved in PRL/GH family, is absent. PLP-L has a putative N-linked glycosylation site (residues 47–50, NMTS).
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FIG. 2. The phylogenic analysis for members of the mouse (m) and rat (r) PRL/GH protein family. Phylogenetic tree construction was performed by Higgins’ method (23) in DNAsis software program. Abbreviations: PRL, prolactin; PLP, PRL-like protein; PRP, PRL-related protein; PL, placental lactogen; PLF, proliferin; PLF-RP, proliferin-related protein; GH, growth hormone.
Of all four new members of PRL family, only PLP-I have a typical secondary structure of PRL family: a large disulfide loop and small loops at both N- and C-termini. The others are devoid of the C-terminal disulfide loop that is conserved in PRL/GH family. Moreover, they have low homology (around 30%) with each other. Therefore, these proteins appear to represent new subclasses of PRL-related hormones, respectively (Fig. 2). Tissue-specific expression of each clone was evaluated by Northern blot analysis of rat tissues (heart, brain, spleen, liver, muscle, kidney, and testis). Only PLP-I (Fig. 3) was detected in the multiple rat tissue Northern blot filter and the other clones were not detected (not shown). As they are derived from placenta cDNA library, their expression may be restricted in placenta as are the case with other PLPs and PLs. Interestingly, PLP-I was detected in testis and not in spleen although it derived from spleen. PLP-I may be expressed in spleen but under the detection level of our Northern blot. The size of PLP-I mRNA was 1.3 kb suggesting the EST clone was almost a full clone with putative 200–300 bp poly A tail. In the literature, the presence of other PRL family members in testis has not been examined although immunoreactive PRL-like protein has been demontrated in rat testis (14). The role played by PRL in male reproduction has been little studied. PRL treatment stimulates testicular growth and spermatogenesis through the direct action on the Leydig cells to increase their ability to bind to LH leading to the enhanced response to gonadotropin stimulation. Recent demonstration of the expression of a pro-
lactin receptor in Sertoli cells and the stimulation of FSH receptors on these cells by PRL expanded the role of PRL in male reproduction (15). However, the fact that the PRL or its receptor knockout mice suffered no male reproductive dysfunction (16–18) suggests that in addition to
FIG. 3. Northern blot analysis of rat multiple tissues (Clontech) with PLP-I probe. Only testis expressed 1.3 kb mRNA of PLP-I.
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PRL, PRL-like proteins may compensate for the loss of PRL. PLP-I may be a good candidate for these proteins. Although there were already fourteen genes for PRL/GH hormones known in rodents, we have identified four more new members of this family. Therefore, the totals of 18 distinct genes for PRL/GH family are now identified in rodents. Whether such a diversity of PRL/GH hormone family is present in human remains to be clarified. Rodents have three isoforms of PRL receptor produced by alternative splicing (short, intermediate, and long forms), while human has only a long isoform. The diversity of ligands may be special for rodents due to this receptor diversity. In fact the binding of PL-I and PL-II to PRL receptor has been shown. However, as PLP-A, PLP-B, PLP-C, PLP-D, PLP-H, and d/tPRP have been shown not to bind to PRL receptors, they may have their specific receptors different from PRL receptors. As PRL receptors and GH receptor are the members of class I cytokine receptor superfamily with recent inclusion of leptin receptor (19), it is possible that the known or unknown cytokine receptors may interact with PLP members. The physiological roles of each new member are remained to be clarified. It will be of considerable interest to determine the spectrum of biological roles of each PLP family members. A recent review on the role of PRL family in regulation of the physiology of pregnancy suggests that these hormones contribute to the broad array of alterations in angiogenesis, hematopoiesis, and lymphocyte function that occur during pregnancy (20). These hormones should be categorized to be as members of cytokine superfamily. In the mean time, our effort to identify the new members of PRL/GH family will continue till the identification of the mammalian homolog(s) of SL. Recent cloning of second isoform of SL in common carp (21) suggests that SL may form a gene family. Our recent lesson from another teleost-specific hormon, stanniocalcin (22), also suggests that we may expect more members of SL in mammals. REFERENCES 1. Goffin, V., Shiverick, K. T., Kelly, P. A., and Martial, J. A. (1996) Endocr. Rev. 17, 385– 410.
2. Bole-Feysolt, C., Goffin, V., Edery, M., Binart, N., and Kelly, P. A. (1998) Endocr. Rev. 19, 225–268. 3. Dai, G., Liu, B., Szpirer, C., Levan, G., Kwok, S. C., and Soares, M. J. (1996) Endocrinology 137, 5009 –5019. 4. Deb, F., Faria, T. N., Roby, K. F., Larsen, D., Kwok, S. C., Talamantes, F., and Soares, M. J. (1991) J. Biol. Chem. 266, 1605–1610. 5. Lin, J., Poole, J., and Linzer, D. I. H. (1997) Endocrinology 138, 5535–5540. 6. Muller, H., Orwig, K. E., and Soares, M. J. (1998) Biochim. Biophys. Acta 1396, 251–258. 7. Iwatsuki, K., Oda, M., Sun, W., Tanaka, S., Ogawa, T., and Shiota, K. (1998) Endocrinology 139, 4976 – 4983. 8. Wilder, E. L., and Linzer, D. I. H. (1986) Mol. Cell. Biol. 6, 3283–3286. 9. Rand-Weaver, M., Noso, T., Muramoto, K., and Kawauchi. H. (1991) Biochemistry 30, 1509 –1515. 10. Steger, R. W., Chandrashekar, V., Zhao, W., Barke, A., and Horseman, N. D. (1998) Endocrinology 139, 3691–3695. 11. Lu., M., Swanson, P., and Renflo, J. L. (1995) Am. J. Physiol. 268, R557–R582. 12. Mousa, M. A., and Mousa, S. A. (1999) Gen. Comp. Endocr. 113, 197–211. 13. Goffin, V., Martial, J. A., and Summers, N. L. (1995) Protein Eng. 8, 1215–1231. 14. Roux, M., Martina, N., Richoux, J.-P., and Grignon, G. (1985) Cell Tissue Res. 240, 663– 667. 15. Guillaumot, P., and Benahmed, M. (1999) Mol. Cell. Endocrinol. 149, 163–168. 16. Horseman, N. D., Zhao, W., Montecino-Rodriguez, E., Tanaka, M., Nakashima, K., Engle, S. J., Smith F., Markoff, E., and Dorshkind, K. (1997) EMBO J. 16, 6926 – 6935. 17. Ormandy, C. J., Camus, A., Barra, J., Damotte, D., Lucas, B. K., Buteau, H., Edery, M., Brousse, N., Babinet, C., Binart, N., and Kelly, P. A. (1997) Genes Dev. 11, 167–178. 18. Steger, R. W., Chandrashekar, V., Bartke, A., and Horseman, N. D. (1998) Endocrinology 139, 3691–3695. 19. Tartaglia, L. A. (1997) J. Biol. Chem. 272, 6093– 6096. 20. Linzer, D. I. H., and Fisher, S. J. (1999) Mol. Endocr. 13, 837– 840. 21. Amemiya, Y., Sogade, Y., Nozaki, M., Takahashi, A., and Kawauchi, H. (1999) Gen. Comp. Endocr. 114, 181–190. 22. Ishibashi, K., Miyamoto, K., Taketani, Y., Morita, K., Takeda, E., Sasaki, S., and Imai, M. (1998) Biochem. Biophys. Res. Commun. 250, 252–258. 23. Higgins, D. G., Bleasby, A. B., and Funchs, R. (1991) CABIOS 8, 189 –191.
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Identification of Four New Members of the Rat Prolactin/Growth Hormone Gene Family Kenichi Ishibashi 1 and Masashi Imai Department of Pharmacology, Jichi Medical School, Tochigi 329-0498, Japan
Received July 14, 1999
The rodent prolactin (PRL)/growth hormone (GH) gene family currently consists of at least 14 distinct genes that are expressed mainly in pituitary, uterus, and/or placenta. We report here the identification of novel four members from rat with significant homology to PRL. The encoding proteins are not homologs of other known members of this hormone family. The four new cDNAs were assigned to PRL family based on sequence homology and were referred to as PRL-like protein-I (PLP-I), PLP-J, PLP-K, and PLP-L, following the current naming order of rodent PLP family, where PLP-H is the most recent gene. They encode amino acids with 211–228 amino acids, and 34 –38% identity with PRL. All have one or two N-linked glycosylation sites. Among the examined rat tissues by Northern blot analysis, only PLP-I was expressed in testis. Our results indicate that the rodent PRL/GH gene family is large with at least 18 distinct genes. © 1999 Academic Press
The pituitary hormones, growth hormone (GH) and prolactin (PRL), and placental lactogen (PL) of placental origin are structurally related and grouped together in the PRL/GH family (1). This family has recently been expanded due to the discovery of rodent genes. These proteins are structurally more similar to PRL (30 – 40%) than to GH (15–21%), suggesting that they may be derived from a PRL gene. Some are named PRL-like peptide (PLP). These rodent proteins are currently made of 18 different members (2). However, some seem to be derived from same gene through alternative splicing or minor strain differences. PLP-C and PLP-Cv are derived from same gene (3). PL-Iv and PL-Imix come from PL-I gene (4). PLP-E and PLP-G are virtually identical (5, 6). Therefore, currently the PRL/GH family consists of 14 distinct genes. These genes are GH, PRL, PLP-A, PLP-B, PLP-C, PLP-D, PLP-E, PLP-F (5) PLP-H (7), PL-I, PL-II, d/tPRL (decidual PRL-related protein), PLF (proliferin) (8), and 1
To whom correspondence and reprint requests should be addressed. Fax: 181-285-44-5541. E-mail: [email protected].
PLF-RP (proliferin-related peptide). Within the PLP subfamily, PLP-C, PLP-D, d/tPRL, and PLP-H are highly homologous. PLP-H is the most recently identified member (7). Recently, a novel member of this family has been discovered in fish pituitary and named somatolactin (SL) (9, 10). SL is similarly related to GH and PRL. The function of SL is not well understood, although the roles in sexual maturation stress responses and calcium and acid base regulation have been suggested. SL has a stimulatory effect on the renal net phosphate reabsorption in renal tubule primary culture (11). Currently, SL is only identified in teleost fishes. SL may be present in all vertebrate species. Moreover, the expression of SL is not limited to pituitary gland. It is also expressed in brain and widely distributed in most brain lesion (12). The structural similarity of SL to the PRL/GH family and the wide distribution in the fish brain prompted us to search for its mammalian homologs in the expressed sequence tag (EST) banks where many clones from mammalian brain cDNAs are deposited. However, we could not identify any mammalian SL homologs. Instead, we identified four more new members of this gene family. Interestingly, one of them was expressed in testis. MATERIALS AND METHODS To identify the new members of PRL/GH family, queries were made in the public dbEST database (maintained at NCBI/NIH) with the coding region of the rat PRL/GH sequences using the BLAST program. Several EST sequences were identified which have amino acid sequences related to PRL that had not been previously reported. Inclusion in the PRL family is dependent upon the positioning of four core cysteine residues and some other key amino acids. Four distinct closely related novel ESTs homologs to PRL were identified. These clones were obtained from Research Genetics (Huntsville, AL), Genome Systems (St. Louis, MO), and American Type Culture Collection (Manassas, VA). These clones were sequenced fully on both strands using fluorescent dye terminator chemistry (Applied Biosystems). Analyses of the nucleotide and amino acid sequences were performed using DNAsis v3.5 (Hitachi Software Engineering Co., Tokyo). Rat multiple tissue Northern blots were purchased from Clontech Laboratories (Palo Alto, CA) which had 2 mg polyA(1) RNA at each
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0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Vol. 262, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Multiple alignments of rat PRL (Prolactin), GH (Growth Hormone), and four new members of PLPs (prolactin-like proteins). The six cysteine residues that form a large disulfide loop and small loops near the amino- and carboxy-termini are indicated by V and X, respectively. The junctions between signal peptides and mature peptides are indicated by Y for PRL and by inverted v for GH.
lane. The membranes were hybridized in ExpressHyb hybridization solution (Clontech) for 3 h at 68°C with probes, randomly 32P-labeled full inserts of each clone containing its open reading frame. The membranes were washed in a final buffer of 0.13 SSC and 0.1% SDS, then exposed to X-ray film at 280°C between two intensifying screens. The blots were reprobed after deprobing in boiled 0.1% SDS solution for 15 min.
RESULTS AND DISCUSSION We identified four new members of PRL family proteins, which are not the currently known members of this family. They have relatively low homology with PRL up to 38%. The most recent new members of this family are PLP-H (7). Following this naming order, we tentatively named these proteins PLP-I, PLP-J, PLP-K, and PLP-L and deposited in GenBank under the accession numbers AB019791, AB019945, AB022882, and AB002283, respectively. The derived amino acid sequences were aligned in Fig. 1. PLP-I was derived from rat spleen cDNA(AA851934) of 1035 nucleotides encoding 220 amino acid residues with a 59-UTR of 241 bp, 39-UTR of134 bp, and polyA tail. A signal peptide seemed to be absent at N-terminus. The open reading frame is 34% identical
with PRL. The 13 cysteine residues were present in PLP-I and four of them can be aligned at the same position as the cysteines in PRL of other vertebrates (indicated by V and X in Fig. 1). These four cysteines will form a large disulfide loop and a small loop near the carboxy-terminus. These are one of the hallmarks of proteins in the PRL/GH family (13), while PRL has an additional small amino-terminal disulfide loop (indicated by X in Fig. 1). The presence of six cysteines in the first 35 amino acid residues of N-terminus is noteworthy. Some of them will form a small N-terminal disulfide loop after the cleavage of a putative signal peptide. PLP-I has a putative N-linked glycosylation site (residues 76-79, NSST). PLP-J was derived from rat placenta cDNA (AA875114) of 810 nucleotides encoding 211 amino acid residues with a 59-UTR of 49 bp, 39-UTR of 128 bp, and polyA tail. The open reading frame is 35% identical with PRL. The first 29 amino acid residues may be a signal peptide (boundary was indicated by Y in Fig. 1) and the mature peptide (182 amino acids) has five cysteines. Two of them will form a large disulfide loop. However, the other cysteines are not conserved. These cysteines may form covalent intramolecular interactions that are not typically found in this family. Therefore, PLP-J is unique in that it lacks both N-terminal and C-terminal small disulfide loops, which is unusual in PRL/GH family. Based on this unique secondary structure, the receptor for PLP-J may be different from that of PRL. PLP-J has two putative N-linked glycosylation sites (residues 77– 80, NNTC and residues 173–176, NKSI). PLP-K was derived from rat placenta cDNA (AI059879) of 842 nucleotides encoding 228 amino acid residues with a 59-UTR of 44 bp, 39-UTR of113 bp, and polyA tail. The open reading frame is 38% identical with PRL. The first 31 amino acid residues may be a signal peptide and the mature peptide (197 amino acids) has five cysteines. Two of them will form a large disulfide loop. As both N- and C-termini have unpaired cysteines, two small disulfide loops at both ends may not be formed. Therefore, the secondary structure of PLP-K may be similar to that of PLP-J. PLP-K has a putative N-linked glycosylation site (residues 173–176, NDTH). PLP-L was derived from rat placenta cDNA (AI028934) of 844 nucleotides encoding 228 amino acid residues with a 59-UTR of 50 bp, 39-UTR of109 bp, and polyA tail. The open reading frame is 37% identical with PRL. The first 27 amino acid residues may be a signal peptide and the mature peptide (201 amino acids) has five cysteines. Four of them will form a large disulfide loop and an N-terminal small loop. However, the C-terminal disulfide small loop, which is conserved in PRL/GH family, is absent. PLP-L has a putative N-linked glycosylation site (residues 47–50, NMTS).
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FIG. 2. The phylogenic analysis for members of the mouse (m) and rat (r) PRL/GH protein family. Phylogenetic tree construction was performed by Higgins’ method (23) in DNAsis software program. Abbreviations: PRL, prolactin; PLP, PRL-like protein; PRP, PRL-related protein; PL, placental lactogen; PLF, proliferin; PLF-RP, proliferin-related protein; GH, growth hormone.
Of all four new members of PRL family, only PLP-I have a typical secondary structure of PRL family: a large disulfide loop and small loops at both N- and C-termini. The others are devoid of the C-terminal disulfide loop that is conserved in PRL/GH family. Moreover, they have low homology (around 30%) with each other. Therefore, these proteins appear to represent new subclasses of PRL-related hormones, respectively (Fig. 2). Tissue-specific expression of each clone was evaluated by Northern blot analysis of rat tissues (heart, brain, spleen, liver, muscle, kidney, and testis). Only PLP-I (Fig. 3) was detected in the multiple rat tissue Northern blot filter and the other clones were not detected (not shown). As they are derived from placenta cDNA library, their expression may be restricted in placenta as are the case with other PLPs and PLs. Interestingly, PLP-I was detected in testis and not in spleen although it derived from spleen. PLP-I may be expressed in spleen but under the detection level of our Northern blot. The size of PLP-I mRNA was 1.3 kb suggesting the EST clone was almost a full clone with putative 200–300 bp poly A tail. In the literature, the presence of other PRL family members in testis has not been examined although immunoreactive PRL-like protein has been demontrated in rat testis (14). The role played by PRL in male reproduction has been little studied. PRL treatment stimulates testicular growth and spermatogenesis through the direct action on the Leydig cells to increase their ability to bind to LH leading to the enhanced response to gonadotropin stimulation. Recent demonstration of the expression of a pro-
lactin receptor in Sertoli cells and the stimulation of FSH receptors on these cells by PRL expanded the role of PRL in male reproduction (15). However, the fact that the PRL or its receptor knockout mice suffered no male reproductive dysfunction (16–18) suggests that in addition to
FIG. 3. Northern blot analysis of rat multiple tissues (Clontech) with PLP-I probe. Only testis expressed 1.3 kb mRNA of PLP-I.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PRL, PRL-like proteins may compensate for the loss of PRL. PLP-I may be a good candidate for these proteins. Although there were already fourteen genes for PRL/GH hormones known in rodents, we have identified four more new members of this family. Therefore, the totals of 18 distinct genes for PRL/GH family are now identified in rodents. Whether such a diversity of PRL/GH hormone family is present in human remains to be clarified. Rodents have three isoforms of PRL receptor produced by alternative splicing (short, intermediate, and long forms), while human has only a long isoform. The diversity of ligands may be special for rodents due to this receptor diversity. In fact the binding of PL-I and PL-II to PRL receptor has been shown. However, as PLP-A, PLP-B, PLP-C, PLP-D, PLP-H, and d/tPRP have been shown not to bind to PRL receptors, they may have their specific receptors different from PRL receptors. As PRL receptors and GH receptor are the members of class I cytokine receptor superfamily with recent inclusion of leptin receptor (19), it is possible that the known or unknown cytokine receptors may interact with PLP members. The physiological roles of each new member are remained to be clarified. It will be of considerable interest to determine the spectrum of biological roles of each PLP family members. A recent review on the role of PRL family in regulation of the physiology of pregnancy suggests that these hormones contribute to the broad array of alterations in angiogenesis, hematopoiesis, and lymphocyte function that occur during pregnancy (20). These hormones should be categorized to be as members of cytokine superfamily. In the mean time, our effort to identify the new members of PRL/GH family will continue till the identification of the mammalian homolog(s) of SL. Recent cloning of second isoform of SL in common carp (21) suggests that SL may form a gene family. Our recent lesson from another teleost-specific hormon, stanniocalcin (22), also suggests that we may expect more members of SL in mammals. REFERENCES 1. Goffin, V., Shiverick, K. T., Kelly, P. A., and Martial, J. A. (1996) Endocr. Rev. 17, 385– 410.
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