Sequence Similarities between a Novel Putative G Protein-Coupled Receptor and Na+/Ca2+ Exchangers Define a Cation Binding Domain
Heli Nikkila1,
D. Randy McMillan,
Brian S. Nunez2,
Leigh Pascoe3,
Kathleen M. Curnow4 and
Perrin C. White
Division of Pediatric Endocrinology University of Texas
Southwestern Medical Center Dallas, Texas 75235-9063
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ABSTRACT
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cDNA clones encoding a novel putative G
protein-coupled receptor have been characterized. The receptor is
widely expressed in normal solid tissues. Consisting of 1967 amino acid
residues, this receptor is one of the largest known and is therefore
referred to as a very large G protein-coupled receptor, or VLGR1. It is
most closely related to the secretin family of G protein-coupled
receptors based on similarity of the sequences of its transmembrane
segments. As demonstrated by cell surface labeling with a biotin
derivative, the recombinant protein is expressed on the surface of
transfected mammalian cells. Whereas several other recently described
receptors in this family also have large extracellular domains, the
large extracellular domain of VLGR1 has a unique structure. It has nine
imperfectly repeated units that are rich in acidic residues and are
spaced at intervals of approximately 120 amino acid residues. These
repeats resemble the regulatory domains of
Na+/Ca2+ exchangers as
well as a component of an extracellular aggregation factor of marine
sponges. Bacterial fusion proteins containing two or four repeats
specifically bind 45Ca in overlay experiments;
binding is competed poorly by Mg2+ but competed
well by neomycin, Al3+, and
Gd3+. These results define a consensus cation
binding motif employed in several widely divergent types of proteins.
The ligand for VLGR1, its function, and the signaling pathway(s) it
employs remain to be defined.
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INTRODUCTION
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In addition to its role as an intracellular messenger,
Ca2+ mediates many interactions between proteins
both inside and outside the cell. Accordingly, many protein domains
have evolved to bind Ca2+ and, in some cases,
other cations. Some of these domains are found in a wide variety of
proteins. Such domains include EF-hands (1), cadherin repeats,
Ca2+ binding epidermal growth factor (EGF)-like
repeats, and thrombospondin repeats (2).
Na+/Ca2+ exchangers consist
of five and six transmembrane domains at the N and C termini,
respectively, separated by a large cytoplasmic loop. This loop contains
two domains spaced 120130 residues apart that act cooperatively to
bind Ca2+ and regulate activity of the exchanger
(3, 4). We now describe a novel putative G protein-coupled receptor
(GPCR), hereafter referred to as VLGR1 (very large G protein-coupled
receptor-1), with an extracellular domain that is one of the largest
identified thus far, consisting of repeated units that strongly
resemble these motifs and that indeed bind Ca2+
and other cations.
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RESULTS
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Isolation of cDNA Encoding a Putative GPCR
Clone 5A1 encoding part of an unknown protein was isolated
serendipitously in 1986 while attempting to isolate cDNA encoding a
cytochrome P450 enzyme, steroid 11ß-hydroxylase (CYP11B1) (5). It
contained an open reading frame of approximately 3.4 kb that extended
to its 5'-end. There was no strong similarity between the predicted
amino acid sequence of the open reading frame and any sequences present
in databases before 1991. However, a hydropathicity plot (not shown)
revealed seven hydrophobic segments near the C terminus of the
predicted protein. The domain containing these segments was
subsequently found to be similar in sequence to several GPCRs (see
below). Accordingly, we attempted to determine the complete coding
sequence of this putative receptor.
We were unable to isolate longer clones by screening several cDNA
libraries. Clones comprising the full-length cDNA were eventually
isolated using several variants of "anchored" PCR. In several
instances, these techniques were unable to extend the sequence. In each
such instance, the corresponding exon was sequenced from genomic DNA
clones, and an additional antisense RT-PCR primer was synthesized
corresponding to sequences near the 5'-end of the exon.
Genomic clones containing portions of the VLGR1 gene were isolated by
hybridization screening of a bacteriophage-
library. Several yeast
artificial chromosome "Mega-YAC" clones were isolated from the CEPH
library (6) by PCR screening. These included clones 851C7, 930A4,
943F7, and 944B4.
Chimerism of the full-length cDNA was ruled out by amplification of
overlapping segments by RT-PCR covering the entire length of the cDNA
(Figs. 1
and 2
) and by mapping of both ends of the
full-length cDNA to the same "Mega-YAC" genomic clones. We
concluded that we had obtained the full-length cDNA because the
sequence encoded a Met residue in an adequate context for translation
initiation with in-frame stop codons 5' of it, because further rounds
of anchored PCR terminated at the same few nucleotides (not shown) and
because RT-PCR using primers corresponding to genomic DNA sequences 5'
of this region failed to yield a product. There were no putative splice
acceptors in the genomic sequence in the region of the putative
transcriptional start site. The full-length cDNA was 6,503 bp,
consisting of 284 and 318 bp of 5'- and 3'-untranslated sequences,
respectively, and an open reading frame of 5,901 bp. It was predicted
to encode a protein of 1,967 amino acid residues (Figs. 1
and 3b
.
Because this protein was predicted to be a very large GPCR, it was
tentatively termed VLGR1 pending functional studies.

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Figure 2. RT-PCRs Covering the Entire Length of VLGR1 cDNA
Lanes are marked as diagrammed in Fig. 1 . Size standards are in base
pairs. RT-minus control reactions showed no amplification (not shown).
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Figure 3. Amino Acid Sequence of VLGR1 (Single Letter Code)
Predicted from the cDNA Nucleotide Sequence (GenBank Accession Number
AF055084)
In the amino acid sequence, the putative N-terminal signal peptide and
transmembrane domains are marked by double underlines,
potential N-linked glycosylation sites are indicated by
shaded N's, and regions similar to
Na+/Ca2+ exchangers are underlined.
A potential palmitoylation site in the C-terminal cytoplasmic domain is
enclosed by a dotted box, and potentially phosphorylated
serine residues in the C-terminal domain are boxed. In
the nucleotide sequence, locations of primers used for PCR are
underlined and numbered with arbitrary
laboratory identifiers; refer to the schematic in Fig. 1 . A
polyadenylation signal is double underlined.
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Figure 3B. Continued underlined.
A potential palmitoylation site in the C-terminal cytoplasmic domain is
enclosed by a dotted box, and potentially phosphorylated
serine residues in the C-terminal domain are boxed. In
the nucleotide sequence, locations of primers used for PCR are
underlined and numbered with arbitrary
laboratory identifiers; refer to the schematic in Fig. 1 . A
polyadenylation signal is double underlined.
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Tissue Distribution of Expression
Hybridization of the original VLGR1 clone to a Northern blot of
total adrenal or testis RNA revealed a faint smear from approximately
4,000 to 7,000 nucleotides after prolonged exposure of the
autoradiogram (not shown); no signal was detected in liver RNA.
Although the original clone contained a poly(A) tail, poly(A)+ mRNA
isolated on oligo(dT) cellulose was not enriched in VLGR1 transcripts.
Possibly the mRNA is relatively unstable. The 320-nucleotide
3'-untranslated region is relatively short and A+U rich (70%), and A+U
rich elements often decrease mRNA stability by promoting deadenylation
(7). This possibility has not actually been studied for VLGR1 mRNA.
The low abundance of VLGR1 transcripts was confirmed by failure to
isolate any additional clones after screening 106
clones from a human adrenal cDNA library, and by isolating only one
shorter VLGR1 clone from 106 clones of a human
testis cDNA library.
Therefore, the tissue distribution of expression was determined by
RT-PCR of total RNA from normal human tissues (Fig. 4
). RNA quantity and quality were
confirmed by simultaneously amplifying a segment of ß2-microglobulin,
a ubiquitous component of class I transplantation antigens (8).
Aliquots were removed every five cycles to ensure sampling during the
exponential phase of amplification. These experiments revealed that
VLGR1 is widely expressed in normal human tissues with the exception of
liver, spleen, and leukocytes (not included in Fig. 4
). In general, a
good signal was detected with VLGR1 primers after 30 cycles of
amplification, whereas ß2-microglobulin primers required
approximately 20 cycles to yield a similar signal. This suggests that
VLGR1 is expressed at low abundance compared with ß2-microglobulin,
which represents approximately 0.01% of total mRNA (8).

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Figure 4. Distribution of VLGR1 Expression in Normal Human
Tissues, Determined by RT-PCR and Visualized by Bromide Staining of
Agarose Gels
The upper band (570-bp PCR product) in each lane
represents VLGR1 expression, whereas the lower band (400-bp
product) represents ß2-microglobulin mRNA. 1, Ovary; 2, small
intestine; 3, gastric mucosa; 4, liver; 5, adrenal; 6, testis; 7,
colon; 8, lung; 9, thyroid; 10, skeletal muscle; 11, foreskin
fibroblasts; 12, kidney; 13, spleen.
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The broad distribution of expression of VLGR1 was confirmed by a search
of the Genbank and expressed sequence tag (EST) databases, which
yielded hits to clones from adrenal adenoma (AA604143 and
AA602663), ductal breast tumor (AA575832), and normal kidney
(AA910218), brain (R88809 and KIAA0686), and retina (W27007).
Chromosomal Localization
All four YAC clones carrying VLGR1 have segments mapping to
chromosome 5q14.1, although several of these clones are chimeric. Based
on linkage markers on these clones, VLGR1 probably maps between D5S618
and D5S1452. Close linkage (16 cR,
560 kb) to D5S618 was confirmed
by radiation hybrid mapping using the Stanford G-3 panel (9) (not
shown). VLGR1 was also mapped to this chromosomal region by fluorescent
in situ hybridization of metaphase chromosomes (10) using
labeled bacteriophage-
clones as probes (not shown).
Expression of VLGR1 Extracellular Domain Fusion Proteins
Bacterial fusion proteins were expressed that contained either
four (pET-24/BD69) of the putative extracellular repeat domains (see
Discussion) or two such domains (pET-24/BD89), in each
case along with T7 antigenic tag sequences. Production of the
appropriate sized fusion proteins (51 and 24 kDa, respectively) was
confirmed by Western blot analysis using anti-T7 antiserum (Fig. 5b
). When nitrocellulose blots of total
bacterial lysates were overlaid with
45CaCl2, bands
corresponding to the fusion proteins were observed only in lysates that
had been induced with
isopropyl-ß-D-thiogalactopyranoside (IPTG)
(Fig. 5a
). No bands corresponding to the fusion proteins were seen in
lanes containing total protein from uninduced cells, from cells
transformed with empty pET-24 vector, or from cells transformed with a
pET vector encoding a protein not known to bind
Ca2+, 11ß-hydroxysteroid dehydrogenase (11).
VLGR1-thioredoxin fusion proteins expressed from pET-32 constructs
(pET-32 encodes different antigenic tags than pET-24) gave results
similar to those seen with the pET-24 fusion proteins (not shown).

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Figure 5. Expression of Bacterial Fusion Proteins
All panels are arranged identically. Each pair of lanes represents
bacteria transformed with the same plasmid before (-) and 2 h
after (+) induction with IPTG. pET24, Empty vector; BD69, pET24
encoding VLGR1 repeat domains 69; BD89, a similar plasmid encoding
repeat domains 89; 11-HSD, pET25 encoding the kidney isozyme of
11ß-hydroxysteroid dehydrogenase, used here as a negative
control; trop, 5 µg of troponin. The positions of mol wt standards
are marked on the left of each panel, and arrows
on the right denote the expected mobilities of each protein. A,
45Ca overlay experiment. B, Western analysis of the same
blot used in panel A. The primary antiserum was horseradish peroxidase
(HRP)-conjugated anti-T7 tag for the left of the panel,
and anti-HSV tag for the right. C, Coomassie blue
staining of an identical gel to confirm protein loading.
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To determine whether other cations could compete with
Ca2+ for binding to VLGR1 fusion proteins,
varying concentrations of unlabeled Ca2+ or other
cations were added to the 45Ca overlays of BD89
protein (Fig. 6
). The other cations
selected were known agonists for the parathyroid gland
Ca2+-sensing receptor (12) and/or for a putative
osteoblast cation-sensing receptor (13). Five millimolar
Mg2+ reduced the background without noticeably
affecting binding to the fusion protein and was added to all
incubations. Gd3+ and neomycin strongly competed
binding of 45Ca, with IC50
values (concentration at which band intensity was reduced 50%) of
approximately 10 µM. Al3+ competed
less well, with an IC50 of 0.3 mM.
Ca2+ itself had an IC50 of
approximately 10 mM.

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Figure 6. Competition of 45Ca Binding by Other
Cations
Intensity of binding by BD89 fusion protein is measured relative to
binding in the presence of 5 mM Mg2+, which is
set equal to 1.0. Points represent one of duplicate
experiments. The log10 of the concentration of each ion is
plotted on the X axis (in µM; e.g. 0 is 1
µM and 3 is 1 mM).
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Expression in Cultured Mammalian Cells
Despite repeated attempts using both peptides and bacterial fusion
proteins, we were unable to raise an antiserum to the extracellular
domain of VLGR1 that recognized the protein in mammalian cells (not
shown). To ensure that the cDNA was translated in vivo into
a full-length protein that was expressed on the cell surface, a segment
encoding an influenza hemagglutin antigenic tag was ligated between
codons 6768 after the region encoding the putative signal peptide.
The resulting cDNA was transiently transfected into 293 human embryonic
kidney cells, and cell lysates were subjected to Western blotting with
an antihemagglutin monoclonal antibody. As shown in Fig. 7
, a band of approximately 220 kDa was
seen only in extracts from cells transfected with this construct and
not in cells transfected with control constructs. In addition, a
fainter smaller band was observed that might correspond to the putative
extracellular domain of the protein; proteolytic cleavage of large
extracellular domains of GPCRs (e.g. TSH receptor) has been
previously observed but is of unknown function (14). Alternatively, the
smaller band might represent underglycosylated protein.

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Figure 7. Cell Surface Expression of VLGR1
A, Western blot of detergent lysate of human embryonic kidney 293 cells
transfected with a control plasmid encoding green fluorescent protein
(lane 1) or with a plasmid encoding HA-tagged VLGR1 (lane 2). Size
standards in kilodaltons are shown. B, Cell surface labeling
experiment. Lane 1, Lysate of 293 cells transfected with the plasmid
encoding HA-tagged VLGR1 (different transfection than lane A2); lane 2,
material bound by streptavidin beads incubated with this cell lysate;
lane 3, supernatant of lysate after removal of beads; lane 4, lysate of
identically transfected cells that had been incubated with
sulfosuccinimidyl-6-(biotinamido) hexanoate before lysis; lane 5,
material bound by streptavidin beads incubated with this cell lysate;
lane 6, supernatant of lysate after removal of beads. Lanes 1, 3, 4,
and 6 contain equal volumes of lysate, and lanes 2 and 5 contain equal
volumes of eluate from the beads.
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Transfected cells were then surface labeled with a biotin
derivative, sulfosuccinimidyl-6-(biotinamido) hexanoate. After
biotinylation, the proteins corresponding to both bands were bound by
streptavidin beads, whereas the beads did not bind unbiotinylated
protein. Thus, the recombinant protein is present at the cell
surface.
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DISCUSSION
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The cDNA Encodes a Novel Putative GPCR
There were two regions in which the coding sequence of VLGR1 is
similar to other nonredundant database sequences as determined by a
gapped BLAST search (15). The region with statistically stronger
sequence similarity to other database entries spanned residues
14001800 in VLGR1, in which there were 78 hits with E values (the E
value is the number of chance hits at a given degree of similarity
expected in the entire database) of <0.00001. All such sequences were
members of a new subfamily of putative GPCRs with large extracellular
domains. More than 50 of these were variants of the
Ca2+-independent latrotoxin receptor
(latrophilin), which is known to be coupled to
G
o (16, 17). A ligand is known for only one
other of these relatively similar putative receptors: CD97 is the
receptor for decay accelerating factor (CD55), a cell surface protein
that inhibits the complement cascade (18). To determine whether these
similarities involved common motifs, the PSI-BLAST program (15) was
used to construct an alignment of those sequences with E values <
0.00001, and then scan the database for sequences similar to the
alignment. One hundred fifty-two new sequences were identified with E
values of 10-5 to10-82, all of
which were known G protein-coupled peptide hormone receptors in the
secretin family (family 2).
The observed sequence similarities (Fig. 8
) cluster in seven hydrophobic domains
of approximately 20 residues each which represent putative
transmembrane regions. In addition, there are completely conserved
cysteine residues just before the first, third, and fifth transmembrane
domains (i.e. they are predicted to be on the extracellular
side of the receptor), at least two of which might form sulfhydryl
bonds stabilizing the correct conformation of the receptor. Based on
these sequence similarities, it appears very likely that VLGR1 is
indeed a GPCR. However, G protein coupling of family 2 GPCRs has not
been studied in detail, and, consequently, amino acid residues of VLGR1
that might be directly involved in signal transduction cannot be
identified at present.

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Figure 8. Alignment of the Predicted Amino Acid Sequence of
VLGR1 with Several Other GPCRs in the Region Encoding
Seven-Transmembrane Domains
The receptors in the alignment are: 1, VLGR1; 2, a human receptor
"similar to D. melanogaster cadherin-related tumor
suppressor" (GenBank locus 1665821); 3, latrophilin (16 ); 4, CD97
(18 ); 5, BAI1(brain angiogenesis inhibitor-1)(41 );6, human vasoactive
intestinal peptide receptor (28 ). Positions that are identical or
highly similar (the accepted similarities are L/V/I, S/T, F/Y, and K/R)
to a consensus of at least four of the six proteins are
boxed. Beginning residues of each protein segment on
each line are numbered; the lines represent continuous
alignments. Transmembrane domains of VLGR1 are denoted by
numbered bold horizontal lines; putative transmembrane
domains for the other proteins are similiarly but not necessarily
identically placed. Three completely conserved cysteine residues that
might participate in sulfhydryl bridges are marked by
triangles.
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The first 23 residues of VLGR1 are predominantly hydrophobic and are
predicted by the PSORT program (19) to form an uncleaved signal peptide
that permits translocation of the nascent peptide across the
endoplasmic reticulum. In contrast, two downstream methionine residues
(M52 and M80) are not followed by putative signal peptides although
they are in adequate nucleotide contexts for initiation of translation.
This supports the identification of M1 as the actual N terminus of the
protein. The large N-terminal domain has 21 potential N-linked
glycosylation sites (N-X-S/T, Fig. 3
), consistent with an extracellular
location. The expression of epitope-tagged recombinant protein on the
cell surface is further evidence that it contains all necessary amino
acid sequences for proper processing.
Near the beginning of the putative C-terminal cytoplasmic domain are
two successive cysteine residues that lie within a potential
palmitoylation sequence. Many other GPCRs have similarly placed single
or paired cysteine residues that are palmitoylated. This type of
covalent modification may affect interactions between the C-terminal
domain and the inner cell membrane and thus modulate accessibility of
the C-terminal domain to phosphorylation (20). Indeed, several high
probability sites for serine phosphorylation exist in the C-terminal
cytoplasmic tail of VLGR1 (Fig. 3
) (21). In other receptors,
phosphorylation of serine residues in the C-terminal cytoplasmic domain
is involved in desensitization after agonist stimulation (22, 23).
Finally, the C-terminal residues correspond to the consensus motif
(Ser/Thr)-Xaa-(Val/Ile/Leu) recognized by proteins bearing so-called
PDZ domains (24). Such proteins, which recognize distinct sequences at
the C termini of target proteins, are believed to act as scaffolds for
assembling signal transduction proteins into functional signaling units
(25). Several GPCRs, such as metabotropic glutamate receptors, are
recognized by proteins with PDZ domains (26). Some, but not all, GPCRs
in family 2 have potential C-terminal recognition sequences for PDZ
domains including receptors for calcitonin (27), PTH, and vasoactive
intestinal peptide (28), as well as CD97 (29), but whether any of these
is functional remains to be determined.
The Putative Extracellular Domain Has a Repetitious Structure
Resembling Na+/Ca2+
Exchangers
The second region of VLGR1 that was similar to existing database
entries spanned residues 301280. Twenty-three similar sequences with
E values of 10-2 to 10-4
were identified, all but one of which were
Na+/Ca2+ exchangers (30).
The region of the exchangers to which VLGR1 was similar was located in
the large cytoplasmic loop and consisted of two acidic domains spaced
120130 residues apart (Fig. 9
). The
other protein with significant similarity to VLGR1 was the large
N-terminal segment of the core protein of the aggregation factor of the
marine sponge, Microciona prolifera (31). To identify
additional related proteins, one iteration of PSI-BLAST was run using
all proteins with E values < 0.001. Fifty-four additional
proteins with E values < 10-3 were
identified, most of which were additional
Na+/Ca2+ exchangers. Ten,
however, represented a portion of the cytoplasmic domain of integrin
ß4 (32) with E values of 10-2 to
10-5, and two were to hypothetical proteins from
Synechocystis sp. Sequence similarities between
Na+/Ca2+ exchangers,
integrin ß4, and the Synechocystis proteins have been
previously noted (33).

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Figure 9. Amino Acid Similarities of VLGR1 Repeat
Domains
Top, Alignment of portions of VLGR1 with each other, and
(below the bold line) with segments of other proteins.
Residues identical or highly similar (as in Fig. 8 ) to a consensus of
at least six sequences are boxed. Starting amino acid
residues of each segment are numbered; note that the
distance between successive segments of VLGR1 is approximately 121
residues or a multiple thereof (bold numbers represent
the position of the initial residue of each segment divided by 121).
Cytoplasmic domains in Na+/Ca2+ exchangers are
from humans (H1, H2), squid (Loligo opalescens,
L1, L2) and fruit flies (D. melanogaster, D1,D2); note
that the distance between each pair of segments averages 125 residues.
Iß4 denotes part of the cytoplasmic domain of integrin ß4. AF3
denotes the second of 14 repeated segments from the MAFp3C aggregation
factor of Microciona prolifera; the average distance
between repeat units in this protein is 117 residues. Pairwise
alignments can be extended beyond the illustrated region but are not
shown for reasons of clarity. Bottom, Schematics
of VLGR1, a Na+/Ca2+ exchanger, integrin ß4,
and MAFp3C aggregation factor. The first three are transmembrane
proteins, and the cell membrane is illustrated by the pair of
thin dotted lines; the outside of the cell is toward the
top. The thick lines represent the
polypeptide chain; they are not drawn to scale. The serpentine portions
of VLGR1 and the Na+/Ca2+ exchanger represent
transmembrane domains. MAFp3C is an extracellular protein that
undergoes proteolytic cleavage (denoted by the broken
line). The repeat units aligned in the top part of the
figure are denoted by numbered circles. Repeats
4 and 7 in VLGR1 cannot be readily aligned with the others although the
120 residue spacing is preserved; the same is true for repeats 7, 10,
and 13 of MAFp3C. In the VLGR1 schematic, S-S denotes a putative
sulfhydryl bond formed between conserved cysteines indicated in Fig. 5 .
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A comparison of the sequence of the putative VLGR1 extracellular domain
with itself revealed clusters of self-identity spaced at intervals of
approximately 120 amino acid residues or multiples thereof. Repeat
units 1, 2, 3, 6, 7, 8, and 9 were most readily aligned (Fig. 9
). The
most highly conserved positions in each repeat were concentrated in an
acidic core of 45 residues. To determine whether this core sequence was
a motif shared with
Na+/Ca2+ exchangers, the
database was searched with PSI-BLAST using 45 residues from VLGR1
repeat unit 6. A weak match (E = 0.26) was identified in the first
iteration to a domain from a squid (L. opalescens)
Na+/Ca2+ exchanger, but the
second iteration identified 40 additional
Na+/Ca2+ exchangers from a
wide variety of vertebrates and invertebrates with E values of
10-2 to 10-7; two domains spaced
120130 residues apart were identified in almost every protein.
These domains are equivalent to the Calx-ß motifs previously
described for these proteins (33).
The similarity between the repeats in VLGR1 and those in
Na+/Ca2+ exchangers extends
to the predicted secondary structure; within the 45-residue core, most
of the repeats have a ßß
ß structure with similar positioning
of the ß-strands in each repeat (not shown). Given the similarities
of sequence and of spacing (120130 residues) between repeats in VLGR1
and Na+/Ca2+ exchangers, it
is reasonable to speculate that these domains have analogous
functions.
In the cardiac sarcolemmal
Na+/Ca2+ exchanger, the
first of the acidic domains strongly bound 45Ca
in overlay experiments, and mutations or deletions of the acidic
residues markedly decreased Ca2+ binding.
Moreover, these mutations, or deletions of the second conserved domain,
abolished regulation of the exchanger by intracellular
Ca2+ (3, 4). Thus, if the repeats in VLGR1 were
truly related to those seen in
Na+/Ca2+ exchangers, they
might bind Ca2+ or other cations. This proved to
be the case in 45Ca overlay experiments under
conditions very similar to those in which domains of the
Na+/Ca2+ exchanger were
studied.
Possible Functions of VLGR1
For those GPCRs with large extracellular domains [e.g.
the LH receptor (34)], the extracellular domain is itself often able
to bind ligand independently of the rest of the protein. Because
portions of the VLGR1 extracellular domain bind
Ca2+ and other cations, one obvious possibility
is that VLGR1 functions as a sensor for cations.
One GPCR is already known to respond to Ca2+: the
Ca2+ sensor of the parathyroid gland (PCaR) (12).
This receptor also has a large extracellular domain with clusters of
acidic residues, but it is most similar to metabotropic glutamate
receptors, and it has no sequence similarity to VLGR1.
Some physiological responses to extracellular
Ca2+ cannot be readily ascribed to the PCaR, and
thus the existence of additional Ca2+ sensing
receptors has been inferred. For example, cultured osteoblasts do not
express the PCaR, yet they respond to extracellular
Ca2+, Al3+,
Gd3+, or neomycin, but not
Mg2+, with increased DNA synthesis (13). These
cations are identical to those that compete 45Ca
binding by VLGR1 fusion proteins in the overlay experiments. However,
VLGR1 was not detected in an osteoblast cell line that is calcium
responsive (not shown), and thus it is a poor candidate for the
osteoblast calcium receptor. Although VLGR1 fusion proteins strongly
bind cations, we speculate that VLGR1 is not a receptor for cations
per se but instead binds its physiological ligand through
Ca2+-mediated interactions. This is plausible
considering that two relatively closely related receptors with other
calcium binding motifs in their ectodomains have large protein
ligandslatrotoxin receptor for latrotoxin (16) and CD97 for
decay-accelerating factor (18). Intriguingly, the organization of the
ectodomain of VLGR1 most closely resembles the N-terminal segment of
the main protein of aggregation factor of the marine sponge,
Microciona prolifera. This highly polymorphic proteoglycan
mediates species-specific cell aggregation. Although the role of the
repeat domains in assembly and function of aggregation factor remains
to be elucidated, this protein provides precedents for the existence of
Calx-ß motifs in extracellular proteins and for the apparent role of
these motifs in protein-protein interactions.
Potential protein interaction partners for the extracellular domain of
VLGR1 might be detected by pulldown experiments or "far-Westerns"
with the bacterial fusion proteins used in the present study, but it is
not yet clear which tissues would represent the best source of protein
for such experiments. Whereas many GPCRs are expressed in a
tissue-specific manner consistent with their functions, VLGR1 is widely
expressed albeit at low levels in solid tissues with the exception of
liver and spleen, in which expression is undetectable. It is not yet
known whether expression might be higher in discrete structures within
particular tissues, or at specific times during development.
In summary, the similarities between the repeats in
Na+/Ca2+ exchangers and
those in VLGR1 expand the functional roles of the Calx-ß cation
binding motif. The presence of repeated domains with related structures
and functions in such widely divergent gene superfamilies as cation
exchangers, sponge aggregation factors, integrins, and GPCRs makes it
tempting to speculate that, as with other consensus
Ca2+ binding motifs, similar domains will be
found in additional proteins of diverse types and functions.
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MATERIALS AND METHODS
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Isolation of cDNA
An oriented human adrenal cDNA library (35) in the plasmid
vector pcD1, was a gift from David Russell (University of Texas
Southwestern, Dallas). Hybridization screening with a part length cDNA
containing part of the 3'-untranslated region of bovine steroid
11ß-hydroxylase (CYP11B1) was performed as described (5), except that
filters were washed at 52 C in 0.075 M NaCl, 0.0075
M Na citrate and 0.05% SDS.
Additional 5'-cDNA sequences were obtained by 5'-RACE (rapid
amplification of cDNA ends, Life Technologies, Inc.,
Gaithersburg, MD) or anchored PCR protocols and mRNA from human thyroid
or from the prostate carcinoma line, LnCAP. Specific primers for each
RACE step (Fig. 1
and Table 1
) were
synthesized based upon already known cDNA or genomic sequences. A
confirmatory 5'-RACE was performed using specific primer 2835.
Isolation of Genomic Clones
Human and murine genomic libraries in bacteriophage Lambda FIX
or Lambda DASH (Stratagene, La Jolla, CA) were screened by
hybridization with VLGR1 cDNA.
The CEPH Mega-YAC library (provided by Denis LePaslier, Institut Jean
Dausset, Paris, France) was screened by PCR using primer pairs
1061A-1066, 10122025, and 139-2224. Vectorette libraries used to
extend the 5'-end sequence of VLGR1 were constructed using
HaeIII, RsaI, and AluI digests of DNA
from YAC 944b4 that were ligated to the annealed vectorette primers
2762 and 2763. They were screened by PCR as described (36) using
vectorette PCR primers 2764 or 2793 and specific VRGR1 primers.
Radiation Hybrid Mapping
Eighty-three DNA samples from the G-3 Stanford Human Genome
Center panel (Research Genetics, Inc., Huntsville, AL)
were amplified using primers W5187 and 2791 (Figs. 1
and 3
).
Reactions were carried out in Epicentre (Madison, WI) buffer J and 35
cycles of 94 C x 30 sec, 55 C x 30 sec, and 72 C x 2
min. Results were analyzed on the Stanford Human Genome Center server
(URL: shgc.stanford.edu/Mapping/rh/search.html) (9).
RT-PCR
Normal human tissues were obtained from the Memorial
Sloan-Kettering Cancer Center Tissue Procurement Service (New York,
NY). RNA was prepared using the acid guanidinium-phenol-chloroform
method (RNAzol, Biotecx, Houston, TX) (37). Semiquantitative
RT-PCR on human samples was performed using random hexamers to prime
cDNA synthesis, and VLGR1 primers 305 and 322 or ß2-microglobulin
primers 393 and 394 (Table 1
). PCR cycle conditions were 94 C x 5
min, at which point Taq polymerase was added, 94 C x 1
min, 60 C x 1 min, and 72 C x 40 sec with an additional 10
sec per cycle added at 72 C. Aliquots were withdrawn at 20, 25, and 30
cycles and subjected to electrophoresis in agarose gels.
To confirm the integrity of the assembled cDNA sequence, overlapping
segments (Figs. 1
and 3
) were amplified by RT-PCR. In all cases, a step
down protocol was used with annealing temperatures lowered from 60 C to
45 C in 1 C increments, followed by 94 C x 15 sec, 45 C x
30 sec, and 72 C x 60 sec, for 30 cycles. Primer pairs were
selected to amplify across introns, so that the corresponding PCR
products using genomic DNA were either of different sizes or failed to
amplify under the same conditions (not shown). Moreover, RT-minus
controls also failed to amplify (not shown).
Construction of Full-Length cDNA
The insert of clone 5A1 was digested with BamHI and
XbaI and subcloned into pBluescriptKS+
(Stratagene). A 2.4 kb segment of additional 5' sequence
was amplified from random primed cDNA derived from LNCaP mRNA in two
segments using primers 2819 and 2862 or W5185 and 2224. PCR
conditions were 95 C x 5 min, at which time Taq
polymerase was added; then 35 cycles of 95 C x 1 min, 50 C
x 1 min, and 72 C x 3 min with an additional 5 sec per cycle at
72 C. In addition, 3% formamide was added to the W5185/2224 PCR. The
isolated fragments were annealed and then amplified with primers 2836
(this primer adds an NcoI site surrounding the initial ATG
and an XbaI site in the 5'-untranslated region) and 2224
using Pfu polymerase (Stratagene) and Pfu buffer I with
the following conditions: 95 C x 2 min followed by 30 cycles of
95 C x 1 min, 40 C x 1 min, and 75 C x 2 min with an
additional 15 sec per cycle added at 75 C. The amplified fragment was
sequenced, digested with XbaI, and ligated to the
XbaI-digested 5A1 subclone. Constructs were verified by
complete sequencing.
Expression of VLGR1 Extracellular Domain Fusion Proteins
Segments of cDNA encoding extracellular repeat units 69
(BD69, amino acid residues 706-1132, see Discussion) or
89 (BD89, residues 940-1132) were amplified by PCR of full-length
cDNA using sense primers BD6S (6, 7, 8, 9) or BD8S (8, 9) and antisense
primer BD9AS. PCR products were ligated into the corresponding sites of
the pET-24a+ vector (Novagen, Madison, WI).
Cultures (50 ml) of BL21(DE3)pLysS cells containing either the
BD69/pET-24 or BD89/pET-24 constructs were grown to an
OD600 of 0.6 and induced with 1
mM IPTG. Cells were collected after 2 h by
centrifugation at 2000 x g and frozen at -20 C. After
thawing, cells were resuspended in SDS-PAGE loading buffer and
sonicated three times using a Branson 200 cell disruptor.
Calcium Overlays
45Ca overlays were performed exactly as
described (38), except that proteins were resolved by electrophoresis
on a 12% SDS-polyacrylamide gel. For binding competition experiments,
blots of lysates of bacteria transformed with BD89/pET-24 were cut
into individual strips and incubated for 10 min in buffer containing 1
µCi/liter 45CaCl2, 5
mM of MgCl2, and varying
concentrations of CaCl2,
GdCl3, AlCl3, or neomycin
sulfate (Sigma, St. Louis, MO). All strips were then
washed (38), air dried and exposed to autoradiography film for 17
days. Band intensities were quantitated using an Eagle-Eye II video
imaging device (Stratagene).
Western blots were washed and probed using enhanced chemiluminescence
reagents (Amersham Pharmacia Biotech, Arlington Heights,
IL) according to the manufacturers protocol.
Expression of Epitope-Tagged VLGR1 in Mammalian Cells
An epitope-tagged construct of VLGR1 was produced by inserting a
54-bp fragment of the influenza virus hemagglutinin (HA1) gene (39)
into the unique AccI site near the 5'-end of the VLGR1 open
reading frame. This construct was subcloned into pCI (Promega Corp., Madison, WI) to produce a mammalian expression cassette
under the control of the cytomegalovirus (CMV) promoter.
The HA-tagged VLGR1 expression plasmid and a control construct encoding
green fluorescent protein under control of the CMV promoter
(CLONTECH Laboratories, Inc. Palo Alto, CA) were
individually transfected into human embryonic kidney (HEK) 293 cells
with Fugene 6 (Roche Molecular Biochemicals, Indianapolis,
IN). Twenty-four hours after transfection, the cells were washed with
ice-cold PBS, gently flushed off the plate with PBS, and collected by
centrifugation. The cells were lysed in 0.5% NP-40, 150 mM
NaCl, 10 mM Tris-HCl, pH 8.0, and debris was pelleted.
Samples were boiled for 5 min in sample buffer (50 mM
Tris-HCl, pH 8.0, 10% SDS, 4 M urea, 12% glycerol, 2%
2-mercaptoethanol).
Cell surface protein biotinylation and subsequent precipitation were
performed as described (40). Twenty-four hours after transfection, HEK
293 cells from one 6-well culture plate were washed with PBS plus 1.0
mM MgCl2, 0.1 mM
CaCl2 and harvested. The cells were resuspended
in 2.0 ml PBS with 1.0 mg/ml sulfosuccinimidyl-6-(biotinamido)
hexanoate (EZ-Link Sulfo-NHS-LC-Biotin, Pierce Chemical Co., Rockford, IL) and incubated on ice for 1 h with
occasional shaking. The biotin-labeled cells were washed twice with PBS
+ 100 mM glycine, resuspended in the same buffer, and
incubated on ice for 30 min to quench the unreacted biotin. The cells
were then collected by centrifugation and stored at -20 C.
Cells were lysed by the addition of 400 µl of 0.5% NP-40 lysis
buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl,
0.5% NP-40) containing 1/100 volume Protease Inhibitor Cocktail
(Sigma) and the cellular debris pelleted. An equal volume
of 2x RIPA (radioimmunoprecipitation assay) buffer (200 mM
Tris-HCl, pH 7.4, 300 mM NaCl, 2 mM EDTA, 2%
TX-100, 2% Na deoxycholate, 0.2% SDS) was added to the supernatant.
One hundred microliters of Ultralink Immobilized Streptavidin
(Pierce Chemical Co.) were added, and the reaction was
incubated at room temperature for 1.5 h with constant shaking. The
beads were washed three times in RIPA + 500 mM NaCl and
three times in RIPA to remove nonspecifically bound proteins. The bound
proteins were eluted by boiling for 5 min in 100 µl of sample
buffer.
Solubilized proteins from each experiment were fractionated on 5%
SDS-polyacrylamide gels and electroblotted to Hybond ECL nitrocellulose
(Amersham Pharmacia Biotech). Western blots were probed
with a mouse monoclonal antibody, HA.11 (BabCO, Richmond, CA), an
alkaline peroxidase-labeled antimouse H+L secondary antibody
(Vector Laboratories, Inc. Burlingame, CA), and enhanced
chemiluminescence reagents.
 |
ACKNOWLEDGMENTS
|
---|
We wish to acknowledge the contributions of Streamson Chua and
Carlos Bacino in isolating the first cDNA clones of VLGR1. We thank
David Russell for a human adrenal cDNA library, Denis LePaslier (Centre
dEtude du Polymorphisme Humaine) for his assistance in
identifying YAC clones, David Ward (Yale University, New Haven, CT) for
assistance with fluorescent in situ hybridization, and
Michael Si for technical assistance. Portions of this work were
carried out while several of the authors were at Cornell University
Medical College (New York, NY).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Perrin C. White, Division of Pediatric Endocrinology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas Texas 75235-9063. E-mail:
pwhit2{at}mednet.swmed.edu
This work was supported by a grant from the Childrens Research
Foundation of the Childrens Medical Center of Dallas. H.N., L.P., and
K.M.C. were supported by fellowships from the American Philosophical
Society, the Charles Revson Foundation, and the Klosk Foundation,
respectively. P.C.W. is supported by the Audry Newman Rapoport
Distinguished Chair in Pediatric Endocrinology at University of Texas
Southwestern Medical Center.
1 Current address, Azusa Pacific University, Azusa, California. 
2 Current address, Louisiana State University, Baton Rouge,
Louisiana. 
3 Current address, Fondation Jean Dausset, Centre dEtude du
Polymorphisme Humaine, 75010 Paris, France. 
4 Current address, Pharmacia Corporation, Sydney, New South Wales,
Australia. 
Received for publication February 21, 2000.
Revision received May 10, 2000.
Accepted for publication May 23, 2000.
 |
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