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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{lambda} 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. 1Go and 2Go) 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. 1Go and 3bGoGo. Because this protein was predicted to be a very large GPCR, it was tentatively termed VLGR1 pending functional studies.



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Figure 1. Schematic of VLGR1 cDNA

The cDNA is represented by the thick horizontal line drawn above a scale marked in kilobases; the slightly thinner sections at the ends represent 5'- and 3'-untranslated regions (see Fig. 3Go). Circles above the cDNA mark locations of regions encoding domains that resemble regulatory domains of Na+/Ca2+ exchangers (see Figs. 3Go and 9Go); the shaded circles denote where repeats are predicted based on conserved spacing but the sequence cannot be aligned with the other repeats. The jagged line represents the region encoding the seven-transmembrane domains of a GPCR (Figs. 3Go and 8Go). The first set of horizontal bars below the cDNA schematic denotes segments used to assemble the complete cDNA sequence. 5'-RACE steps are denoted by the specific antisense primer used for each reaction (Table 1Go) and a left arrow. Genomic segments are denoted by gray bars; the pairs of numbers below each bar represent primer pairs that permit the segment to be amplified from genomic DNA (Table 1Go). The second set of horizontal bars represents RT-PCRs used to demonstrate expression of the entire predicted cDNA. The numbers in circles correspond to the lanes in Fig. 2Go; the pairs of numbers below each bar represent primer pairs (see Fig. 3Go).

 


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Figure 2. RT-PCRs Covering the Entire Length of VLGR1 cDNA

Lanes are marked as diagrammed in Fig. 1Go. 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. 1Go. 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. 1Go. A polyadenylation signal is double underlined.

 
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. 4Go). 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. 4Go). 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.

 
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-{lambda} 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. 5bGo). 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. 5aGo). 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; BD6–9, pET24 encoding VLGR1 repeat domains 6–9; BD8–9, a similar plasmid encoding repeat domains 8–9; 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.

 
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 BD8–9 protein (Fig. 6Go). 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 BD8–9 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).

 
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 67–68 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. 7Go, 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.

 
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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1400–1800 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{alpha}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. 8Go) 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.

 
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. 3Go), 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. 3Go) (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. 9Go). 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. 8Go) 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. 5Go.

 
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. 9Go). 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 ßß{alpha}ß 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go and Table 1Go) were synthesized based upon already known cDNA or genomic sequences. A confirmatory 5'-RACE was performed using specific primer 2835.


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Table 1. Oligonucleotides Used

 
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, 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. 1Go and 3Go). 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 1Go). 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. 1Go and 3Go) 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.


    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 d’Etude 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 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. Back

2 Current address, Louisiana State University, Baton Rouge, Louisiana. Back

3 Current address, Fondation Jean Dausset, Centre d’Etude du Polymorphisme Humaine, 75010 Paris, France. Back

4 Current address, Pharmacia Corporation, Sydney, New South Wales, Australia. Back

Received for publication February 21, 2000. Revision received May 10, 2000. Accepted for publication May 23, 2000.


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