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
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ABSTRACT
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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.
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INTRODUCTION
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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.
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RESULTS
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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. 1
, 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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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.
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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. 2A
) 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 2540% and 2025%, 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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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).
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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. 2C
, 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
, AC). 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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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.
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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. 4A
). 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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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).
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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 4C57 region, respectively (Fig. 5
, 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
4C57 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).
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DISCUSSION
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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 (
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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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 140159)
and part of the putative A domain (amino acids 119138) 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 Bouins
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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