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Division of Pediatric Endocrinology University of Texas Southwestern Medical Center Dallas, Texas 75235-9063
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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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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 120–130 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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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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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/BD6–9) of the putative extracellular repeat domains (see
Discussion) or two such domains (pET-24/BD8–9), 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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). 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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, 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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), 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 30–1280. 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
120–130 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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). 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
120–130 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 ßßß structure with similar positioning
of the ß-strands in each repeat (not shown). Given the similarities
of sequence and of spacing (120–130 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 ligands—latrotoxin 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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The CEPH Mega-YAC library (provided by Denis LePaslier, Institut Jean Dausset, Paris, France) was screened by PCR using primer pairs 1061A-1066, 1012–2025, 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 W518–7 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 W518–5 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 W518–5/2224 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 6–9
(BD6–9, amino acid residues 706-1132, see Discussion) or
8–9 (BD8–9, 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
BD6–9/pET-24 or BD8–9/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 BD8–9/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 1–7
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 manufacturer’s 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by a grant from the Children’s Research Foundation of the Children’s 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 d’Etude 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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-Latrotoxin receptor, latrophilin, is a
novel member of the secretin family of G protein-coupled receptors.
J Biol Chem 272:21504–21508-Latrotoxin receptor CIRL/latrophilin 1 (CL1) defines an unusual
family of ubiquitous G-protein-linked receptors. G-protein coupling not
required for triggering exocytosis. J Biol Chem 273:32715–32724This article has been cited by other articles:
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 D. R. McMillan, K. M. Kayes-Wandover, J. A. Richardson, and P. C. White Very Large G Protein-coupled Receptor-1, the Largest Known Cell Surface Protein, Is Highly Expressed in the Developing Central Nervous System J. Biol. Chem., January 4, 2002; 277(1): 785 - 792. [Abstract] [Full Text]
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