Cloning of Two Novel Mammalian Paralogs of Relaxin/Insulin Family Proteins and Their Expression in Testis and Kidney

Sheau Yu Hsu

Division of Reproductive Biology Department of Gynecology and Obstetrics Stanford University School of Medicine Stanford, California 94305-5317


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Based on sequence homology to insulin and relaxin, we have isolated two novel genes of the insulin superfamily from mouse tissues. Because these proteins show a high similarity to relaxin and relaxin-like factor (RLF or Ley I-L), they were named as RIF1 (relaxin/insulin-like factor 1) and RIF2 (relaxin/insulin-like factor 2). After RT-PCR, full-length cDNAs of RIF1 and RIF2 were obtained from mouse testis and ovary, respectively. In addition, a putative human ortholog of RIF1 was isolated from human testis. The deduced coding regions of mRIF1, mRIF2, and hRIF1 were 191, 145, and 213 amino acids, respectively, and all three proteins contain a typical signal sequence for secretion at their amino terminus. Sequence comparison indicated that RIFs encode proteins consisting of B and A subunits connected by a long C domain peptide, and the deduced mature proteins of these putative ligands are most closely related to relaxin, RLF, and insulin from different species. Northern blot analysis showed that RIF1 transcripts are approximately 1.2 kb in size and are expressed mainly in testis of mouse and human. In contrast, RIF2 message of 2.0 and 1.2 kb are preferentially expressed in mouse kidney and are lower in testis, heart, and brain. In addition, immunohistochemical analysis showed that testis expression of RIF1 is restricted to interstitial cells surrounding seminiferous tubules. In kidney, the RIF2 message is localized to selected epithelial cells of loop of Henle. The exclusive expression pattern of RIF1 and related RLF in testis interstitial cells suggested potential physiological roles of these two distinct insulin/relaxin family ligands in testis function. Additionally, the spatial expression pattern of RIF2 suggests a novel role of RIF2 in nephrophysiology. Identification of RIF polypeptides expands the family of relaxin- and insulin-like hormones and allows future elucidation of the physiological role and hormonal mechanisms for these tissue-specific factors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In multicellular organisms, intercellular communication frequently depends on secreted peptide hormones and growth factors acting on neighboring or distant target cells. Insulin, insulin-like growth factors I and II (IGF-I and -II), and relaxin belong to an ancient family of peptide hormones evolved to serve diverse physiological roles ranging from carbohydrate metabolism and organ growth to reproductive functions (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Recent studies have shown that the mammalian insulin family proteins consist of at least six members including insulin, IGF-I, IGF-II, and relaxin, together with a relaxin-like peptide of Leydig cell origin [relaxin-like factor (RLF) or Ley I-L] and an early placenta insulin-like peptide (EPIL or INSL4) (2, 11, 12). Although the overall sequence identity among family members is low, all family proteins share a characteristic B-C-A domain structure in which the B and A domains of mature proteins are covalently linked by two interdomain disulfide bonds. In addition, an additional intradomain disulfide bond within the A domain has been shown to be important for the proper folding and function of these proteins.

Insulin and IGFs are essential growth factors with multiple biological activities, including mitogenesis, differentiation, and angiogenesis of diverse cell lineages. They function by stimulating the autophosphorylation of their receptors and downstream signaling pathways (4, 8, 13, 14, 15). Relaxin, which is produced mainly by reproductive tissues, was first identified in 1926 as a factor that causes relaxation and softening of the pubic ligaments (16). Subsequently, relaxin was found to act on diverse tissues including the reproductive tract, heart, mammary gland, and brain; however, its putative receptor has not been identified (3, 17, 18, 19, 20, 21, 22, 23, 24). The recently identified RLF and EPIL have been shown to be mainly produced by gonads and placenta, respectively. However, their physiological functions remain unclear. While insulin and IGF-I receptors are known to mediate the actions of insulin and IGFs, a tissue-specific orphan receptor (IRR: insulin receptor-related receptor) has been identified (25, 26), suggesting the existence of additional ligands belonging to the insulin/relaxin superfamily.

Recent sequencing of expressed sequence tags (EST) has led to the availability of nucleotide sequence information for up to half of all human genes (27). Consequently, the EST database in the GenBank has become a valuable resource for identifying novel paralogs of known genes in the same species and orthologs from different species. Based on amino acid sequence homology in the putative B and/or A domains of insulin and relaxin, we have identified two novel members of the insulin/relaxin hormone family [relaxin/insulin-like factor 1 and 2 (RIF1 and RIF2)] and generated specific anti-RIF antibodies. Protein sequences deduced from RIF cDNAs indicated an overall similarity in the domain arrangement found in insulin and relaxin. The novel expression pattern of RIFs detected by Northern hybridization and immunohistochemical studies suggests that RIF1 could be involved in the regulation of testis function, whereas RIF2 may have a role in kidney function. Identification of these novel insulin/relaxin superfamily members and generation of anti-RIF antibodies allows the characterization of the endogenous proteins and elucidation of their physiological roles.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of RIFs and Comparison of RIF Sequences with Other Genes in the Insulin/Relaxin Gene Family
Using the BLAST program, we identified three sets of overlapping ESTs encoding motifs with similarity to the B and/or A domains of insulin and relaxin from different vertebrates. These overlapping ESTs were used to identify open reading frames (ORFs) encoded by these novel genes. Using mRNA isolated from mouse ovary and mouse testis as the templates for the synthesis of the first-strand cDNA and subsequent rapid amplification of cDNA ends (RACE)-PCR, cDNAs containing the entire ORF for these two contigs of mouse origin were obtained (mRIF1 and mRIF2, Fig. 1Go, A and B). Full-length cDNA corresponding to the third contig of human origin was isolated from human testis (hRIF1, Fig. 1CGo). In addition, sequence analysis of 5'-RACE products of the second mouse contig cDNAs (mRIF2) showed that there are two variant transcripts of this gene which differ in their 5'-untranslated regions and the ATG start site (Fig. 1BGo). The long form contained an extra stretch of 10 amino acids at the N terminus. A homology search of known genes in the available databases did not reveal any sequences identical to these novel cDNAs. Because sequence comparison with all nonredundant GenBank entries based on full-length sequences showed that the deduced ORF of the two novel mouse cDNAs are most closely related to relaxin, RLF and insulin from different species (mRIF1, 26% identity to relaxin; mRIF2, 27% identity to relaxin; hRIF1, 23% identity to relaxin), they were named RIF (relaxin/insulin-like factor). In addition, because the RIF cDNA of human origin showed significant homology to mouse RIF1 ({cong}45% identity and 66% similarity, Fig. 1DGo) across the whole length of ORF and showed a similar tissue-specific expression pattern, it could be the human ortholog of the mouse RIF1 gene and was tentatively termed as human RIF1 (hRIF1).



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Figure 1. Deduced ORF of RIF Polypeptides

Nucleotide and deduced protein sequences of mouse RIF1 (A), mouse RIF2 (B), and human RIF1 (C). Amino acid numbers are on the right and the stop codon is marked with an asterisk. The longest ORF for mRIF1, mRIF2, and hRIF1 predicted polypeptides of 191, 145, and 213 amino acids, respectively. All RIFs are rich in basic residues (mRIF1, 15.7%; mRIF2, 15.9%; hRIF1, 17.8%) and have high pI values. D, Sequence alignment of mRIF1 and hRIF1. Residue numbers are on the left and similar residues are indicated by + between the aligned sequences.

 
The deduced coding regions of mRIF1 and mRIF2 were 191 and 145 amino acids, respectively, whereas the hRIF1 encodes a 213-amino acid polypeptide. All three novel polypeptides contained a typical signal sequence for secretion after the ATG start codon (SignalP Server, http://genome.cbs.dut.dk/htbin/nph-webface) (Fig. 2AGo) and had a basic isoelectric point (mRIF1, 9.34; hRIF1, 9.71; mRIF2, 9.32). Alignment of RIFs with known mouse and human insulin/relaxin family proteins, including IGF-I, IGF-II, insulin, relaxin, RLF, and EPIL, showed that RIF polypeptides contain the classical B-C-A domain configuration present in the insulin/relaxin family proteins, and the similarity among these proteins was restricted to the regions corresponding to the B and A domains found in mature proteins of insulin and relaxin (Fig. 2AGo) (28). The identity of B and A domains between RIFs and relaxin are approximately 25–40% and 20–25%, respectively. In contrast, the putative C domain of these novel polypeptides showed no phylogenetic relatedness with any known gene. Thus, RIFs are similar to insulin and relaxin but differ from IGF-I and IGF-II, in having a long-connecting peptide situated between the B and A chains (Fig. 2BGo).



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Figure 2. Sequence Comparison between RIFs and Other Genes in the Insulin/Relaxin Family

Panel A, B and A domains of RIFs in comparison with IGF-IA, IGF-II, insulin, relaxin, RLF, and EPIL from mouse and human. Putative cleavage sites between RIF domains were derived based on sequence comparisons with other family proteins and consensus cleavage sites for different proteolytic enzymes. Sequence comparisons were optimized based on cysteine alignment. Dark shaded residues represent identity in at least 7 of the 11 aligned sequences shown, whereas lightly shaded residues indicated identity in at least 5 of the 11 aligned polypeptides. Asterisks above aligned sequences indicate residues that have been shown to be critical for the functions of multiple family members. Residue numbers are on the left. Cysteine residues are in bold letters and dashed lines represent the disulfide bond pattern proven for some family proteins. B, Line diagrams showing the relative length of different functional domains in the insulin/relaxin family proteins. C, Phylogenetic relatedness of mammalian insulin/relaxin family proteins with the B-C-A domain arrangement based on analysis of full-length sequences. m, Mouse; h, human. GenBank accession numbers are: mouse IGF-IA (124261), mouse IGF-II (124256), mouse insulin 1 (124511), mouse insulin 2 (124519), mouse relaxin (1350570), mouse RLF (1754739), mouse RIF1 (AF135823), mouse RIF2 (AF054842), human IGF-IA (124260), human IGF-II (124255), human insulin 1 (124617), human EPIL (1220315), human relaxin 1 (132280), human relaxin 2 (132281), human RLF (3851207), human RIF1 (AF135824), chimpanzee insulin (266377), chimpanzee relaxin 1 (1710080), chimpanzee relaxin 2 (1710081), marmoset monkey RLF (3850653), rhesus monkey insulin (223965), rhesus monkey relaxin (132301), bovine RLF (3719459), equine relaxin (2506784), guinea pig relaxin (1710088), pig relaxin (132309), pig RLF (1708498), rat insulin 1 (124512), rat insulin 2 (124520), rat relaxin (132315).

 
The most unique feature of the B and A domains of insulin superfamily proteins (IGF-I, IGF-II, insulin, relaxin, RLF, insect bombyxins and molluscs insulin-like proteins) is the conserved cysteine residues important for disulfide bond formation and the invariant spacing between these cysteine residues. Within the B and A domains, six cysteines (Fig. 2CGo, bold letters) that are critical for disulfide bond formation and the proper folding of insulin-related polypeptides are completely conserved in the three RIFs, suggesting that RIFs are structurally conserved when compared with other family proteins. In addition, the leucine and glycine residues flanking the first cysteine found in the receptor binding site-containing B domain of several members, are conserved in all RIFs (17) (Fig. 2AGo).

The full-length amino acid sequence alignments among different mammalian insulin/relaxin family proteins were used to generate a phylogenetic tree using the Block Maker program, and the mRIF1 and hRIF1 were grouped in a single branch (Fig. 2DGo). While insulin from different mammals form a distinct phylogenetic branch, relaxin, RLF, RIF1, and RIF2 are grouped under a separate branch, suggesting that during evolution RIFs diverged early from other hormones in this family and could have derived from the ancestor gene that also gave rise to relaxin and RLF (Fig. 2DGo).

Expression of RIF mRNA in Multiple Tissues
Analysis of EST sequences in the Genbank showed that ESTs containing partial sequences of mRIF1 and mRIF2 could be found in diverse tissues. Mouse RIF1 is encoded by sequences from mammary gland, thymus, colon, and pooled organ libraries, whereas mouse RIF2 sequences were derived from thymus and colon libraries, suggesting that these tissues could express these proteins. In contrast, ESTs with hRIF1 sequences were derived exclusively from the testis cDNA library. To investigate the expression pattern of RIFs, mRNA from mouse heart, brain, spleen, lung, liver, muscle, kidney, and testis as well as from human spleen, thymus, prostate, testis, uterus, small intestine, colon, and leukocyte, were examined (Fig. 3Go, A–C). Northern blot hybridization analysis showed that the expression of RIF1 in both mouse and human is highly restricted (Fig. 3Go, A and C). A main transcript of 1.2 kb for both mRIF1 and hRIF1 was confined to testis and no obvious signals could be detected in other mouse or human tissues. Thus, the expression profile of hRIF1 closely reflected the distribution profile of corresponding EST in the GenBank. In contrast, the RIF2 mRNA is expressed in multiple tissues including kidney, testis, heart, and brain (Fig. 3BGo). One major transcript with a size of 1.2 kb was detected in these tissues, together with one minor transcript of 2.0 kb found mainly in kidney and testis. These different mRNA species could result from the use of alternative polyadenylation sites and/or alternative splicing of RIF2.



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Figure 3. Expression of RIF mRNA Transcripts in Mouse and Human Tissues

For Northern blot analysis, poly (A)+-selected RNA from different tissues of mouse or human (Tissue blots; CLONTECH Laboratories, Inc.) were hybridized with a 32P-labeled RIF1 or RIF2 cDNA probe. After washing, the blots were exposed to x-ray films at -80 C. Subsequent hybridization with a ß-actin cDNA probe was performed to estimate nucleic acid loading (8 h exposure). Specific transcripts for mRIF1 (A), mRIF2 (B), and hRIF1 (C) are indicated by arrows and RNA size standards are indicated on the left. Hybridization signals for ß-actin are shown at the bottom panels.

 
Generation of RIF Antibodies and Immunohistochemistry
To detect the specific cell lineages expressing RIF1 and RIF2 polypeptides in mouse tissues, we generated rabbit polyclonal anti-RIF1 and anti-RIF2 antibodies using synthetic oligopeptides conjugated to keyhole limpet hemocyanin (KLH). After collection of antiserum, antibody titers were estimated using a peptide-specific enzyme-linked immunoadsorbent assay, and aliquots of antiserum were further purified using affinity columns. Immunohistochemical detection using paraffin-embedded adult mouse tissues showed that RIF1 expression in testis is restricted to the interstitial cells, and no specific signal was evident in germ cells and Sertoli cells within the seminiferous tubules (Fig. 4AGo). In the kidney, immunoreactive RIF2 is detected in selected epithelial cells of loop of Henle (Fig. 4FGo). Under higher magnification, the immunohistochemical staining of RIF1 (Fig. 4BGo) and RIF2 (Fig. 4GGo) is localized in cytoplasm of these positive cells. Negative control staining using preimmune serum (Fig. 4Go, C and H) or presaturated antibodies (Fig. 4Go, D and I) as the primary antibody showed no specific signals. In addition, staining of sections of pancreas, a control tissue, with anti-RIF1 (Fig. 4EGo) or anti-RIF2 (Fig. 4JGo) antibody showed negligible nonspecific staining.



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Figure 4. Immunohistochemical Detection of RIF1 and RIF2 Expression in Testis and Kidney

RIF1 expression in testicular sections using anti-RIF1 antibody C1588 (panel A, x100; and panel B, x200). RIF2 expression in kidney sections using anti-RIF2 antibody C1337 (panel F, x100; and panel G, x200). Specific signals are indicated by arrows. Panels C and H are adjacent sections hybridized with preimmune serum for anti-RIF1 and anti-RIF2 antibodies, respectively. Panels D and I are adjacent sections hybridized with anti-RIF1 and anti-RIF2 antibodies presaturated with antigen peptide, respectively. I, Interstitial cells; S, seminiferous tubules; Tn, thin wall loop of Henle; Tk, thick wall loop of Henle. Panels E and J are pancreas sections hybridized with anti-RIF1 and anti-RIF2 antibodies, respectively (x100).

 
Chromosome Localization
Fluorescent in situ hybridization analysis using bacterial artificial chromosome library-derived genomic fragments showed that mRIF1 and mRIF2 genes are located on mouse chromosome 19C3 region and 4C5–7 region, respectively (Fig. 5Go, A and B). While no other family gene is located on chromosome 4, mouse insulin 1 and relaxin have been assigned to chromosome 19 using microcell hybrids.



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Figure 5. Chromosome Localization of Mouse RIF Genes

Using mouse genomic fragment of mRIF1 or mRIF2 as probe, RIF1 (panel A) and RIF2 (panel B) genes were localized to chromosome 19C3 region and 4C5–7 region, respectively, by the fluorescence in situ hybridization (FISH) method. Denatured chromosomes from synchronous cultures of mouse lymphocytes were hybridized with biotinylated probes for localization. Assignment of the FISH mapping data (left) was achieved by superimposing signals with 4,6-diamdino-2-phenylindole-banded chromosomes. Analyses are summarized in the form of mouse chromosome ideograms (right).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have identified two novel mammalian paralogs of the insulin/relaxin gene family expressed in restricted tissues. RIF1 is found mainly in testicular interstitial cells whereas RIF2 is expressed by multiple tissues. The homology between RIFs and other proteins in this family is predicted to extend from the primary amino acid sequence to the secondary and tertiary structures based on their characteristic functional domain arrangement and six invariant cysteine residues important for protein architecture. These attributes suggest that there is a conserved interchain disulfide linkage in RIFs as found in other family proteins and posttranslational modifications (such as the eventual removal of the putative C domain) could be important for the derivation of functional RIFs. Furthermore, sequence comparison and phylogenetic analysis indicated that RIFs diverged from other insulin/relaxin family members early during evolution and thus are likely serving distinct physiological functions.

Known members of the insulin/relaxin family were originally grouped based on strong structural similarities and similar posttranslational modifications required for the generation of functional peptides (29). Subsequent studies showed that some of these genes also share similar genomic structure, demonstrating close phylogenetic relatedness (30). However, these proteins have diverged greatly in their biological actions. The best characterized member, insulin, produced by pancreatic ß cells, is important in carbohydrate homeostasis, whereas the corpus luteum-derived relaxin regulates myometrial activity and connective tissue remodeling (3, 18, 20). On the other hand, IGF-I and IGF-II are growth factors acting in both a paracrine and endocrine manner (4, 15). They are important for organ-specific growth and overall anabolism. For the two remaining members of this family in mammals, RLF has been shown to enhance relaxin action whereas the physiological role of the placental-derived EPIL remains unknown (2, 31, 32). In invertebrates, there are at least five molluscan insulin-related peptides (9, 10) and more than six families of insect prothoracicotrophic hormone bombyxins (6, 28, 33, 34) sharing structural similarity with mammalian insulin/relaxin family proteins. These insulin/relaxin family members from invertebrates function as neuroendocrine factors important for the regulation of growth as well as protein and carbohydrate metabolism (9, 35, 36).

Although the physiological importance of insulin-related peptides is well recognized, thus far only the receptors for insulin and IGFs have been cloned (8, 14, 37). In contrast, characterization of the putative receptor for relaxin and RLF is limited to ligand binding studies (38, 39). While the receptors for most insulin/relaxin family proteins are still unknown, an orphan receptor IRR sharing a common structure with insulin and IGF receptor has been cloned (25, 26). This finding suggests the existence of additional ligands belonging to the insulin/relaxin protein family in mammalian genomes. Analyses of chimeric receptors consisting of different functional domains of these cloned receptors have provided information on the ligand specificity and tyrosine phosphorylation of these receptors (13, 40). However, the putative ligand for IRR remains elusive (2, 3, 32). At the present time, it is unclear whether RIFs bind to the known insulin and IGF receptors or to the putative receptors for relaxin and RLF. The finding of RIFs and the generation of anti-RIF antibodies would allow future production of functional recombinant RIFs for the characterization of RIF receptors.

The prepropolypeptide structure shared by most members of the insulin/relaxin superfamily consists of a signal peptide and B, C, and A domains. Based on sequence and overall structural comparison, the insulin/relaxin family members can be subdivided into two groups. The first group consists of hormones (IGF-I and IGF-II) with the B and A domains located at the N- and C terminus, respectively, and are joined by a short connecting C domain that is retained in the mature hormone. In contrast, the remaining members of this family contain a long-connecting C domain of varying length that is normally cleaved to give rise to the functional two-subunit peptide linked by interchain disulfide bonds. The C domain sequences diverged greatly among family proteins and showed no similarity to any known proteins. It is possible that this diversity evolved due to the lack of selection pressure in this region. Among the family proteins, RIFs are similar to relaxin, RLF, insulin, and EPIL in having a long-connecting peptide. Of interest, the C domains of mRIF1 and hRIF1 exhibit very basic isoelectric points (pIs) (10.59 and 9.77 for mRIF1 and hRIF1), which is also shared by EPIL (9.99). The prevalence of multiple dibasic residues in this region could serve as alternative proteolytic sites for generating functional variants.

Several putative proteolytic sites are situated closely to the B and A domain of RIFs. At the C terminus of the mRIF2 B domain and the C terminus of the RIF1 C domain, a cluster of basic residues conforms completely to the consensus cleavage sites for furin-like proteinases (Arg-X-Arg/Lys-Arg) (29, 41). Similar proteolytic motifs also exist at the junctions between C and A domains in insulin, EPIL, and relaxin. In addition, a tribasic motif was found in the C terminus of the mRIF2 C domain. These regions are likely to be important for generating mature two-subunit RIF proteins. However, because multiple dibasic proteolytic sites are scattered along the C domain of proRIF polypeptides, the exact structure of mature RIF1 and RIF2 awaits future investigation on native proteins. In our preliminary studies, Western blotting analysis of RIF1 and RIF2 expression in transfected CHO or 293T cells indicated that RIF proteins probably require cell-specific posttranslational machinery for proper processing and folding, as observed for other family proteins (data not shown). Thus, functional study of RIF proteins requires future investigations on the exact proteolytic processing sites of native proteins and the production of recombinant RIF proteins using appropriate cell types that express RIF proteins endogenously.

While B and A domains are better conserved as compared with the C domain among paralogs in the same species, isolation of orthologs from diverse species has demonstrated that the primary sequences of B and A domains have also diverged greatly in relaxin and RLF. For example, the mature human relaxin and RLF shared only 41% and 70% overall identity to their mouse homologs. Likewise, the putative B and A domains of mRIF1 appeared to share low identity ({cong}59% identity) with hRIF1; nonetheless, the similarity between mRIF1 and hRIF1 encompasses the whole molecule when compared with other family genes. One unique feature found only in the A domain of hRIF1 is its extended C terminus, which contains multiple dibasic residues for proteolytic processing and may give this protein altered structural characteristics. Assuming that hRIF1 and mRIF1 are authentic orthologs in mouse and human, RIF1, together with RLF and relaxin, represents one of the least conserved group of polypeptides among known proteins that have an average of 86.4% identity between mouse and human orthologs (42). These results suggest that protein architecture of RIFs is probably more conserved than the primary sequences.

In the mature relaxin protein, a conserved amino acid motif, R-XXX-R, close to the first cysteine of the B domain, has been shown to be important for relaxin binding to its putative receptor in many tissues (17, 28, 43). Of interest, this motif is retained in all vertebrate relaxins including those from the primitive elasmobranches, and alteration of basic residues in this binding motif abolished receptor interaction (3, 17, 18). Although RLF has been shown to interact with specific binding sites in the uterus and brain (38, 44), synthetic RLF also showed low-affinity binding to the relaxin receptor, possibly through an alternative R-XXX-R motif one helix turn downstream from the corresponding receptor-binding region in relaxin (44). While RLF itself showed no relaxin-like activity, RLF significantly enhanced relaxin-mediated widening of the symphysis pubis in mouse, and it has been proposed that cross-talk of ligands between RLF and relaxin could be important for their functions (44). The predicted mature RIF proteins share only 25% identity with relaxin or RLF, and comparison of the region corresponding to the R-XXX-R receptor-binding motif indicated only one conserved arginine residue in each RIF. In addition, no alternative R-XXX-R motif was found in RIFs. These data suggest that RIFs have evolved alternative receptor recognition sites that are different from other members of the insulin/relaxin family.

Immunohistochemical analysis of the expression of the two murine RIF proteins indicated that they are restricted to specific cell types in selected tissues. In testis of adult mice, RIF1 is found exclusively in the interstitial cells, similar to what has been described for RLF, which was originally isolated as a Leydig cell-specific insulin-like gene (31, 45, 46). Although the function of RLF is unclear, the expression of RLF in testicular interstitial cells has been shown to conform with the differentiation status of these cells (46). The discovery of an additional insulin/relaxin family protein in the male gonad suggests that testicular functions could be regulated by an intricate mechanism mediated through these novel ligands. Recent studies indicated that the testis of dogfish shark produces high amounts of immunoreactive relaxin (47). Although the exact identity of the immunoreactive material in elasmobranch is unclear, a testicular signaling pathway mediated by RLFs could have evolved early in vertebrates. In contrast, the finding of RIF2 expression in specific cells of loop of Henle suggest that RIF2 could have a regulatory role in the kidney. Interestingly, relaxin has been shown to act as a potent renal vasodilator, and chronic administration of relaxin reduces plasma osmolality (48, 49). It is, however, important to note that Northern blot analysis and GenBank EST data suggest that mRIF1 and mRIF2 transcripts are also expressed in other tissues. Future studies on the expression pattern of these proteins in different tissues should provide insights regarding their potential targets and physiological roles.

The localization of RIF2 gene to mouse chromosome 4 in which no other family genes are present is consistent with the hypothesis that ancestor genes of the insulin/relaxin superfamily proteins have dispersed throughout the entire genome during evolution. In contrast, the colocalization of RIF1 on mouse chromosome 19 together with relaxin suggests that these two closely related genes may derive from the duplication of an ancestor gene (50). Further investigation on the structure of RIF genes and their physiological roles will shed light on the evolutionary relationship of genes in the insulin/relaxin family and the diversification of their endocrine and paracrine functions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Computational Analysis
cDNA sequences related to insulin and relaxin were identified from the EST database (dbEST) at the National Center for Biotechnology Information using the BLAST and Gapped BLAST server with the BLOSUM62 comparison matrix (51). The alignment of sequences for genes in the insulin/relaxin family was carried out by the IDENTIFY server (http://dna.Stanford.EDU/identify) and Block Maker at Blocks WWW server (http://blocks.fhcrc.org/blocks/blockmkr). The Block Maker program also calculated the branching order of aligned sequences and the phylogenetic relatedness of aligned sequences by the Cobbler and Gibbs algorithms. Analyses of primary and secondary structures of encoded polypeptides were conducted using multiple web servers including BCM search launcher (http://gc.bcm.tmc.edu:8088/search-launcher/launcher.html), Biology Workbench (http://gc.bcm.tmc.edu:8088/search-launcher/launcher.html), Blocks (http://www.blocks.fhcrc.org/blocks/), and ExPASy Molecular Biology Server (http://expasy.hcuge.ch/).

Identification and Isolation of the Full-Length cDNA for mRIF1, mRIF2, and hRIF1
We searched the GenBank for ESTs with homology to insulin and relaxin and identified two clusters of overlapping clones (RIF1: AA240120, AA940549, AA711108, and AA711156; RIF2: AA689027, AA529046, AA764178, and AA119153) from mouse tissues and a unique sequence from human tissue (T19007) encoding novel sequence motifs with homology to the B and/or A domains of insulin and relaxin from diverse species. For the isolation of full-length cDNA fragments, specific primers were designed based on EST sequences and were used to prepare cDNA pools enriched with the candidate cDNAs by utilizing mouse ovary and testis mRNA as templates. Two micrograms of mRNA were reverse transcribed by using 25 U of avian myoblastosis virus reverse transcriptase (AMV RNase) with oligo(dT) primer, 0.5 mM deoxynucleoside triphosphate, and 20 U of ribonuclease inhibitor. After second strand synthesis with T4 DNA polymerase, the enriched cDNA pool was tailed at both ends with adaptor sequences to allow PCR amplification using specific primers. The tailed cDNA products were then employed as a template for amplification of the candidate cDNA using internal primers. For the isolation of the human homolog for RIF1, a Marathon-ready testis cDNA pool (CLONTECH Laboratories, Inc., Palo Alto, CA) was used as the template for 5'- and 3'-RACE with adaptor- and gene-specific primers. All PCR amplifications were performed under highly stringent conditions (annealing temperature >68 C) using Advantage DNA polymerase (CLONTECH Laboratories, Inc.) or Pfu DNA polymerase (Stratagene, San Diego, CA) to minimize mismatching and infidelity during PCR amplification. PCR products were fractionated using agarose electrophoresis, and specific bands showing hybridization with radiolabeled cDNA probes were subcloned into the pUC18 vector (Invitrogen, San Diego, CA) to further identify candidate clones. At least two independent PCR clones were sequenced to verify the authenticity of the coding sequences of each cDNA. The resulting sequences were assembled into a contig using the ClustalW 1.7 at BCM Search Launcher (http://dot.imgen.bcm.tmc.edu:9331/multi-align).

Northern Blot Analysis
For mRNA analysis, blots containing poly(A)+ RNA from various adult mouse or human tissues (CLONTECH Laboratories, Inc.) were hybridized with specific 32P-radiolabeled RIF cDNA probes generated by random priming (Life Technologies, Inc., Gaithersburg, MD) for 2 h in QuickHybri solution (CLONTECH Laboratories, Inc.). After hybridization, membranes were washed at a stringency of 0.2 x SSC, 0.5% SDS at 60 C and exposed to x-ray film at -80 C with two intensifying screens (Amersham Pharmacia Biotech, Buckinghamshire, UK). To estimate mRNA loading, the blots were subsequently hybridized with a ß-actin cDNA probe.

Generation of RIF Antibodies
Synthetic peptides were coupled to KLH, and used to generate rabbit polyclonal anti-RIF antibodies (HTI Bio-Products, Inc., Ramona, CA). The mRIF1 and mRIF2 peptide antigens (RIF1: LQKKSTNKMNTFRSLFWGNH, Ab C1588; RIF2:HSVVSRRDLQALCCREGCSM, Ab C1337) correspond to the C terminus of the putative C domain of mouse RIF1 (amino acids 140–159) and part of the putative A domain (amino acids 119–138) of mouse RIF2. These specific antibodies were further purified using affinity columns, and the titer of resulting fractionations was determined using ELISA against the peptide antigen.

Immunohistochemical Analysis
Mouse tissues were obtained from adult mice after euthanasia with carbon dioxide and embedded in paraffin after fixation in Bouin’s solution or 4% paraformaldehyde. After deparaffin in xylene, tissue sections were blocked with 5% goat serum in PBS for 30 min to saturate nonspecific binding sites. The primary rabbit polyclonal antibody to RIF1 or RIF2 was diluted to 1:400 in PBS containing 5% goat serum. Sections were incubated overnight at 4 C or 2 h at room temperature in a moist chamber and then washed three times for 20 min each in PBS with 0.1% Tween 20. Negative controls were performed in all cases by substituting the primary antibody with rabbit preimmune serum or antibodies presaturated with the peptide antigen. After incubation with the primary antibody, sections were incubated with gold-conjugated goat antirabbit secondary antibody for 20 min at room temperature. Sections were then washed extensively in PBS with 0.1% Tween 20 before being stained with SilvEnhance solution (Zymed Laboratories, Inc., South San Francisco, CA), counterstained with hematoxylin, and mounted with Paramount for examination under bright field microscopy. Micrographs were taken using an optiphot microscope (Nikon, Melville, NY).

Isolation of Genomic Clones and Identification of Chromosome Localization
To reveal the chromosomal localization of RIF genes from mouse, genomic DNA fragments were isolated from a mouse bacterial artificial chromosome (BAC) genomic library (Genome Systems, St. Louis, MO) using the near full-length RIF1 and RIF2 cDNA probes. Positive BAC clones were digested with various restriction enzymes and confirmed by Southern hybridization. The genomic fragments were then used as probes for fluorescence in situ hybridization (FISH) to mouse metaphase chromosomes (SeeDNA Biotech, Inc., Toronto, Canada). Denatured chromosomes from synchronous cultures of mouse lymphocytes were hybridized with biotinylated probes for signal localization.


    ACKNOWLEDGMENTS
 
I am very grateful to Dr. Aaron J. W. Hsueh for his encouragement and critical review of this paper. I also thank Caren Spencer for editorial assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. Sheau Yu Hsu, Department of Gynecology and Obstetrics, Stanford University School of Medicine, 300 Pasteur Drive, Room A344, Stanford, California 94305-5317.

Received for publication April 1, 1999. Revision received July 28, 1999. Accepted for publication August 20, 1999.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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