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Division of Reproductive Biology Department of Gynecology and Obstetrics Stanford University School of Medicine Stanford, California 94305-5317
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, A
and B). Full-length cDNA corresponding to the third contig of human
origin was isolated from human testis (hRIF1, Fig. 1C). 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. 1B). 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 (
45%
identity and 66% similarity, Fig. 1D) 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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) 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. 2A) (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. 2B).
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, 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. 2A).
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. 2D). 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. 2D).
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. 3, A–C). Northern blot
hybridization analysis showed that the expression of RIF1 in both mouse
and human is highly restricted (Fig. 3, 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. 3B). 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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). In the kidney,
immunoreactive RIF2 is detected in selected epithelial cells of loop of
Henle (Fig. 4F). Under higher magnification, the immunohistochemical
staining of RIF1 (Fig. 4B) and RIF2 (Fig. 4G) is localized in cytoplasm
of these positive cells. Negative control staining using preimmune
serum (Fig. 4, C and H) or presaturated antibodies (Fig. 4, 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. 4E) or
anti-RIF2 (Fig. 4J) antibody showed negligible nonspecific
staining.
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, 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 (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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication April 1, 1999. Revision received July 28, 1999. Accepted for publication August 20, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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