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Genes to Cells (2004) 9, 549-560. doi:10.1111/j.1356-9597.2004.00743.x
© 2004 Blackwell Publishing or its licensors
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DREG, a developmentally regulated G protein-coupled receptor containing two conserved proteolytic cleavage sites

Tetsuo Moriguchi1,a, Keiko Haraguchi1, Naoko Ueda1, Masato Okada2, Toshio Furuya3,4 and Tetsu Akiyama1,*

1 Laboratory of Molecular and Genetic Information, and 3 Laboratory of Molecular Regulation, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
2 Department of Oncogene Research, Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
4 PharmaDesign Inc., 4-2-10 Hacchoubori, Chuou-ku, Tokyo 104-0032, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
We have identified and characterized a novel member of the G protein-coupled receptor (GPCR) family, termed DREG. DREG belongs to the LNB-TM7 subfamily and possesses a long amino-terminus that contains a CUB domain, a PTX domain, a hormone binding domain and a GPCR proteolytic site (GPS) domain. RT-PCR experiments and whole mount in situ hybridization in mice showed that DREG is expressed at high levels in the heart and somite during embryogenesis and in the adult lung. When DREG was transiently expressed in mammalian cultured cells, a 35-kD fragment was generated by endogenous proteolytic processing at the conserved GPS domain. This short fragment was found associated with the cell membrane, typical of GPCRs. DREG was further cleaved in the middle of the extracellular domain, generating a soluble 70-kD fragment containing the CUB and PTX domains. This processing was inhibited by an inhibitor of the endoprotease furin but not of matrix metalloproteinases. These results raise the possibility that DREG plays a role in development, not only as a receptor or an adhesion molecule but also as a secreted ligand.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins that generally act as cell surface receptors responsible for the transduction of a remarkably diverse set of endogenous signals (Bockaert & Pin 1999). Because they participate in almost every known physiological process, GPCRs are the target of ~50% of marketed drugs and represent a major focus in functional genomics programs and drug development research (Ballesteros & Palczewski 2001; George et al. 2002). Although all GPCRs have seven-transmembrane (TM7) segments, these receptors fall into several families that have significant amino acid similarity within but not between families. Family A, also known as family 1, includes the rhodopsins, adrenergic and dopaminergic receptors and receptors for other small organic ligands, whereas family B, also known as family 2, consists mostly of receptors for peptide hormones such as secretin.

A recently defined subdivision within family B, termed LNB-TM7, consists of proteins with long ectodomains composed of functional modules such as epidermal growth factor repeats, cadherin, lectin, laminin, olfactomedin, immunoglobulin or thrombospondin (Stacey et al. 2000). Most of these putative GPCRs are expressed in the brain and immune system, and several of these have apparent functions in development. For example, flamingo is essential for the planar cell polarity in Drosophila, and mutation of Celsr1, which is one of three mammalian homologs of flamingo, disrupts the planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse (Usui et al. 1999; Curtin et al. 2003). The very large G-protein coupled receptor-1 (VLGR1), another member of the LNB-TM7 family, is mutated in the Frings mouse, which is prone to audiogenic seizures (Skradski et al. 2001; McMillan et al. 2002). These results support the notion that some members of the LNB-TM7 subfamily are important for normal development. However, the LNB-TM7 family of proteins is difficult to study because of their large size, genomic complexity and increasing number of member proteins (Fredriksson et al. 2002, 2003).

In the present study, we identified a novel member of the LNB-TM7 family, DREG (for developmentally regulated G protein-coupled receptor), in the human genome, and cloned the full-length mouse and human cDNAs. We show that DREG is expressed at high levels in the heart and somites during mouse embryogenesis and in the adult lung. Furthermore, we demonstrate that DREG is cleaved into a membrane-associated fragment and a soluble fragment at two sites in the extracellular domain; one site is located in the GPS domain and another site is presumably cleaved by a protease of the proprotein convertase family.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Isolation of DREG cDNA

Searching for novel GPCRs in the human genomic database, we identified a human genomic sequence encoding a previously unreported family B-type GPCR localized to chromosome 6. Since the correct splice-sites were unclear, we attempted to identify the coding region from EST sequences and cDNA libraries. By searching the NCBI databases using the human genomic sequence encoding the TM7 region as bait, a mouse EST clone (GENBANK accession number AA796299) containing a sequence homologous to the human genomic sequence was identified. Using oligonucleotide primers derived from this clone, the 5'-end of the mouse cDNA was amplified by PCR, and the full-length cDNA was obtained. To obtain the human cDNA, we performed colony hybridization of a human brain cDNA library, and identified two incomplete cDNA clones. The 5'-end of the human cDNA was amplified from a human keratinocyte cDNA library by PCR. From these sources, several alternatively spliced human transcripts were identified. Alternative splicing that includes or excludes exon 6 distinguishes isoforms 1 and 2 (Fig. 1A and Supplementary material). Alternative splicing that includes or excludes exon 25 changes the usage of termination codons to create the {alpha} and ß isoforms (Fig. 1B and Supplementary material). The truncated carboxy-terminus of the mouse {alpha} isoform is created by the presence of an in-frame termination codon in exon 25 (Fig. 1B). The sequence of the human {alpha}1 isoform encodes a protein of 1221 amino acid residues (Supplementary material). A partial sequence of this gene was recently predicted by Fredriksson et al. (2003). We searched the protein sequence for functional domains using the SMART program, and identified a CUB domain, a PTX domain, a HBD, and a GPS domain (Supplementary material). The first 37 residues of DREG are predominantly hydrophobic and are predicted to form an uncleaved signal peptide.



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  		Figure 1  Schematic representation of the structure of the DREG gene. Genomic organization of the 5'-region (A) and 3'-region (B) of the DREG gene on human chromosome 6 and alignment of splice variants cloned from human and mouse DREG. The alternative splicing that creates the {alpha}, ß, 1, and 2 isoforms of DREG is illustrated. Termination codons (STOP) are indicated by vertical bars.

 
Expression and localization of mouse DREG transcripts

Northern blots of multiple mouse adult tissues were probed with a mouse DREG cDNA probe, but no signal was detected (data not shown). We therefore performed RT-PCR analysis of multiple tissue cDNA panels using two pairs of primers designed from amino-terminal region (CUB +PTX) and 3' UTR, respectively. After 30-cycle PCR using primers from the amino-terminal region, the predicted 948-bp fragment was observed to be expressed at a high level in the lung and at low levels in the heart, spleen and other tissues (Fig. 2). In addition, DREG was highly expressed at embryonic day (E) 11, E15, and E17, but not at E7, indicating that DREG expression is induced between E7 and E11 (Fig. 2). Similar results were obtained when the primers from the 3' UTR were used for PCR amplification (Fig. 2). To examine the spatial and temporal patterns of DREG transcripts, we performed whole mount in situ hybridization experiments. Consistent with the results of RT-PCR, DREG was expressed in the presomite mesoderm at high levels at E9 and in the somite at E10 and E11 (Fig. 3, white arrowheads), but was barely detectable at E7 (data not shown). DREG was also expressed in the heart and developing face, including the frontonasal process and pharyngeal arches (Fig. 3A,B).



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  		Figure 2  Comparison of tissue expression patterns of mDREG mRNA by RT-PCR. Mouse multiple tissue cDNA panels were amplified by PCR with oligonucleotide primers derived from amino-terminus (CUB+PTX) and 3'-untranslated region (3' UTR) of mouse DREG. In a control experiment, PCR reactions were performed with specific oligonucleotide primers for the mouse G3PDH gene. For a negative control, H2O was used instead of cDNA (no sample).

 


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  		Figure 3  DREG mRNA patterns during early embryonic development. Expression of DREG is detected with a probe specific for the extracellular region of mDREG at E9 (A), E10 (B) and E11 (C, anti-sense). DREG is expressed in the presomitic mesoderm at E9 (A, white arrowheads), and in the somite at E10, and E11 (C white arrowheads). Black arrowheads in B and C indicate expression in the developing heart. A result of in situ hybridization with DREG sense probe is shown as a negative control (C sense). Similar results were obtained with another probe specific for a different region of DREG (see Experimental procedures).

 
DREG is cleaved at the GPS domain

By analogy to the other LNB-TM7 receptors (Ponting et al. 1999; Usui et al. 1999; Gray et al. 1996; Krasnoperov et al. 1997; Ichtchenko et al. 1999; Nechiporuk et al. 2001; Abe et al. 2002; Stacey et al. 2002; Obermann et al. 2003), we assumed DREG would be cleaved at the GPS domain, generating two protein bands under standard denaturing SDS-PAGE conditions. We therefore generated two distinct antibodies recognizing the amino-terminus (anti-CUB) and carboxy-terminal cytosolic region (anti-C), respectively. Anti-C antibody recognized a protein of 35-kD from lysates prepared from 293T or COS7 cells that had been transfected with hDREG{alpha}1 (Fig. 4A, lanes 1 and 2), whereas anti-CUB antibody did not recognize any protein (data not shown). Anti-C antibody also recognized a protein of ~60-kD from 293T cells expressing a fusion of GFP to the carboxy-terminus of hDREG{alpha}1 (Fig. 4A, lanes 3 and 4), suggesting that addition of a GFP tag to the carboxy-terminus of hDREG{alpha}1 did not alter its cleavage pattern. Anti-C antibody also detected other isoforms of DREG exogenously expressed in COS7 cells, as shown in Fig. 4B. Since DREG contains more than 1000 amino acids, these results suggest that DREG is cleaved and that only the carboxy-terminal fragment is retained in the cell.



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  		Figure 4  Immunoblotting analysis with anti-DREG antibody. 293T cells (A left panel) and COS7 cells (A right panel, and B) were transiently transfected with each of the expression plasmids indicated. Total cell lysates were resolved by 2–15% SDS-PAGE and subjected to immunoblotting with anti-C polyclonal antibody. Specifically reacted bands are indicated by white arrowheads.

 
The GPS region in many LNB-TM7 receptors, including DREG, contains four conserved Cys residues and other residues (Fig. 5A). Rat CIRL and Ig-Hepta have been reported to be cleaved at the Leu-Thr peptide bond present immediately after the four conserved Cys residues (Krasnoperov et al. 1997; Abe et al. 2002). The GPS domain of DREG contains the sequence His-Phe-Thr, which corresponds to the conserved cleavage site in CIRL and Ig-Hepta, His-Leu-Thr (Fig. 5A). To examine the role of the GPS domain in the cleavage of DREG, we generated several mutants of hDREG{alpha}1, C803S, C822S, C835S and C837S, in which each of the four conserved Cys residues, Cys803, Cys822, Cys835 and Cys837, was replaced with Ser. We also generated the hDREG{alpha}1 mutants T841A and T841P, in which the cleavage site Thr841 was replaced with Ala and Pro, respectively. Each mutant was expressed in COS7 cells and analysed by immunoblotting with anti-C antibody. In contrast with wild-type DREG, none of the mutants examined generated a ~60-kD protein, suggesting that these conserved amino acid residues are required for the proper conformation of the GPS domain and normal proteolytic processing (Fig. 5B). A protein of ~170 kD was detected in lysates from cells expressing C803S, C822S, C835S, C837S, or T841P, and may be the non-cleaved form of the DREG protein, since it was recognized by both anti-C and anti-CUB antibodies (data not shown, also see Fig. 9B, T841P). One mutant, T841A, produced a protein of ~140-kD that reacted with anti-C antibody but not with anti-CUB antibody. This mutant is predicted to be transported to the cell membranes (see below). The identity of the ~140-kD protein will be addressed below.



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  		Figure 5  GPS domain and proteolytic processing. (A) Comparison of the amino acid sequences of the GPS domain among LNB-TM7 proteins. The residues in black are conserved cysteine residues, and the grey boxes indicate highly conserved sequence motifs. (B) COS7 cells were transfected with an expression plasmid for each of the GFP-tagged hDREG{alpha}1 proteins; wild-type (WT), and its point mutants (C803S, C822S, C835S, C837S, T841A, and T841P). Twenty-four hours after transfection, cell lysates were subjected to immunoblotting with anti-C polyclonal antibody. A band of ~140 kD was generated by T841A (white arrowhead, see text).

 


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  		Figure 9  Inhibition of hDREG-T841A processing by small molecule inhibitors. (A) Amino acid sequence alignment around the second cleavage site S2 between human and mouse DREG. The grey boxes indicate highly conserved amino acids, and the proteolytic processing site is shown by the arrow. (B) COS7 cells were transfected with an expression plasmid for each of the GFP-tagged hDREG{alpha}1 mutants; R468A, R468T841AP, R468T841AA, T841A, and T841P. The transfected cells were grown in the presence of the indicated concentrations of a furin inhibitor or MMP inhibitor (GM6001). Twenty-four hours after transfection, cell lysates were subjected to immunoblotting with anti-C antibody (upper panel). The same membrane was re-probed with anti-CUB antibody (lower panel). The ~180-kD, ~170-kD, and ~140-kD proteins are indicated by black arrowheads, white arrowheads, and an arrow, respectively. The asterisk (lower panel) indicates a nonspecific protein.

 
Subcellular localization of DREG and its mutants

We investigated the role of the GPS domain in determining the subcellular localization of DREG by examining the subcellular distribution of carboxy-terminally GFP-tagged hDREG and its mutants with confocal microscopy. Wild-type DREG was detected throughout the cytoplasm, with an intense signal observed in the proximity of the plasma membrane, consistent with the idea that DREG is present in the plasma membrane (Fig. 6A,B). On the other hand, the C803S, C822S, C835S, C837S, and T841P mutants were detected in intracellular vesicles, while the T841A mutant was detected throughout the cytoplasm and weakly in intracellular vesicles (Fig. 6C,D, and data not shown). It has been reported that CIRL and Ig-hepta are cleaved at the GPS domain in the ER (Abe et al. 2002; Krasnoperov et al. 2002), suggesting that the DREG mutants, except for T841A, may have accumulated in the ER lumen and were not transported to the cell surface.



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  		Figure 6  Subcellular localization of DREG. COS7 cells were transfected with an expression plasmid for GFP-tagged wild-type hDREG{alpha}1 (A and B), T841P (C), or T841A (D). The carboxy-terminal region of each protein was detected by anti-GFP antibody. In some cases, wild-type hDREG was localized in the plasma membrane (B, white arrowheads). Scale bars, 10 µm.

 
Determination of the cleavage site in the membrane-proximal region of hDREG

To determine the proteolytic cleavage site in DREG, we constructed a chimeric protein, hWT-Fc, that was composed of the amino-terminal extracellular domain of hDREG{alpha}1 and the Fc domain of human IgG1. When this chimeric construct was expressed in 293T cells, its was proteolytically processed as seen in the above experiments (Fig. 4), and yielded a 36-kD fragment containing the carboxy-terminal Fc portion (Fig. 7A). Amino acid sequencing analysis of this fragment revealed that its amino-terminal sequence is Thr-His-Phe-Gly-Val-Leu-Leu-Glu-Pro-Lys. This indicates that the cleavage site resides between residues Phe840 and Thr841 in the GPS domain (Fig. 7B). As expected, when 293T cells were transfected with a construct, C837-Fc, that was composed of the amino-terminal extracellular domain of hDREG{alpha}1C837S and the Fc region, no cleavage fragment was observed.



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  		Figure 7  Identification of the cleavage site within human DREG. (A) The wild-type hDREG-Fc fusion protein (WT-Fc) and C837S-Fc fusion protein (C837S-Fc) were purified from the culture medium of 293T cells. Purified proteins were subjected to 2–15% SDS-PAGE and visualized by silver staining. Proteins of 130 kD and 36 kD are indicated by an arrow and arrowhead, respectively. (Lower panel) Schematic representation of the 130-kD and 36-kD proteins. (B) Amino acid sequences surrounding the proteolytic cleavage sites present in the hDREG-Fc fusion protein. The amino-terminal residues of 130-kD and 36-kD proteins, determined by Edman degradation, are shaded.

 
Determination of a second cleavage site in hDREG

When 293T cells were transfected with expression constructs for hWT-Fc or C837S-Fc and conditioned media from these cell cultures was analysed, they were found to contain a protein of ~130-kD, which is likely to be the uncleaved full-length fusion protein (Fig. 7A). Unexpectedly, amino acid sequencing analysis of this fragment revealed that its amino-terminal sequence is Ser-Leu-Glu-Asp-Glu-Pro-Arg-Leu-Val-Leu-Trp-Ala-Leu-Leu-Val, indicating that a cleavage site is located between the PTX and HBD domains (termed cleavage site S2, Fig. 7B). Consistent with these results, anti-CUB antibody did not recognize the ~130-kD protein (Fig. 8A). In addition, these results raise the possibility that the ~140 kD protein detected in T841A-transfected cells may be a product of DREG cleaved at the S2 site (see Fig. 5B).



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  		Figure 8  Identification of the cleavage site within mouse DREG. (A) The hDREG-Fc (hWT-Fc) and mDREG-Fc fusion proteins (mWT-Fc) were purified from the culture medium of 293T cells transiently transfected with an expression plasmid for hWT-Fc or mWT-Fc. Purified proteins were subjected to 2–15% SDS-PAGE and visualized by silver staining or by immunoblotting with anti-CUB antibody. Immunoreacted proteins of ~75 kD and ~160 kD are indicated by arrows. Proteins of 120 kD and 36 kD, corresponding the 130-kD and 36-kD proteins in Figure 7, respectively, are indicated by arrowheads. (Lower panel) Schematic representation of the 160-kD, 120-kD, 75-kD and 36-kD proteins. (B) Amino acid sequences surrounding the proteolytic cleavage sites present in mDREG-Fc fusion protein. The amino-terminal residues of 120-kD and 36-kD proteins, determined by Edman degradation, are shaded.

 
Determination of the cleavage sites in mDREG

To confirm the existence of two proteolytic sites in DREG, we used a fusion protein, mWT-Fc, containing the extracellular domain of the mouse DREG and the Fc domain of human IgG1. mWT-Fc was expressed in 293T cells, and the chimeric proteins purified from culture media were subjected to SDS-PAGE followed by silver staining. This analysis revealed four proteins of ~36-kD, ~75-kD, ~120-kD, and ~160-kD (Fig. 8A). Amino acid sequencing analysis of the ~36-kD protein revealed that its amino-terminal sequence is Thr-His-Phe-Gly, which matches with the conserved GPS cleavage site (Fig. 8B). This indicates that mouse DREG is also cleaved at the peptide bond between Phe-Thr in its GPS domain.

To reveal the nature of the other three proteins, the chimeric proteins were subjected to immunoblotting with anti-CUB antibody. The ~75-kD and ~160-kD proteins, but not the ~120-kD protein, reacted with anti-CUB antibody (Fig. 8A). Moreover, the ~75-kD protein was not detected by anti-human Fc antibody (data not shown). These observations suggest that the ~160-kD protein represents the full-length mWT-Fc, and that the ~75-kD protein may be an amino-terminal fragment generated after the second cleavage. Unlike hDREG, mDREG may be only partially cleaved at the S2 site, and the amino-terminal ~75-kD fragment obtained may have been copurified as a result of its association with the full-length mWT-Fc, perhaps through oligomerization via the PTX domain. Amino acid sequencing analysis of the ~120-kD protein revealed that its amino-terminal sequence is Asp-Ile-Met-Asp-Asp-Asp-Lys, which is present in the region corresponding to the S2 site in hDREG (Fig. 8B).

Proteolytic cleavage of DREG by the proprotein convertase family of enzymes

Alignment of human and mouse amino acid sequences around the S2 site revealed that the residues at the cleavage site are partially conserved (Fig. 9A). The four residues Lys-Val-Lys-Arg are conserved between human DREG (ex. amino acids 465–468 in hDREG{alpha}1) and mouse DREG (amino acids 437–440 in mDREG{alpha}2), whereas the first residue after the cleavage site is different; Ser469 in human DREG{alpha}1 and Asp441 in mouse DREG{alpha}2, respectively. To determine the importance of the amino acid residues around the cleavage site for the proteolytic processing, we generated two mutants: R468A-Fc consist of the Fc region of human IgG1 fused to hDREG{alpha}1 containing a Arg468 to Ala mutation, and S469A-Fc is the same with Ser469 mutated to Ala. 293T cells were transfected with plasmids expressing wild-type or either of the two mutant chimeric derivatives, and proteins were purified from medium, fractionated by SDS-PAGE and analysed by silver staining and immunoblotting. In contrast to WT-Fc and S469A-Fc, R468A-Fc generated a large amount of a ~180-kD protein that was detected with anti-CUB antibody, indicating that mutation of Arg468 prevented specific processing of DREG (data not shown). To confirm that cleavage at the S2 site occurred intracellularly, we generated additional three mutants tagged with GFP at their amino-termi: R468A, in which Arg468 of hDREG{alpha}1 was replaced with Ala, R468T841AP, in which Arg468 and Thr841 were replaced with Ala and Pro, respectively, and R468T841AA, in which both Arg468 and Thr841 were replaced with Ala. Mutants were expressed in COS7 cells and analysed by immunoblotting with anti-C and anti-CUB antibodies. Immunoblotting with anti-C antibody detected a fragment of ~60-kD in lysates from R468A-expressing cells, whereas a protein of ~170 kD, similar to T841P, was detected in lysates from R468T841AP-expressing cells (Fig. 9B). These results suggest that processing at the GPS domain occurred independent of, and probably prior to the cleavage at the S2 site. Unlike R468T841AP, R468T841AA generated a protein of ~180-kD, presumably because of a difference in glycosylation (Fig. 9B).

The results of the above experiments led us to re-examine the sequences of these proteins, and we noticed that the cleavage site sequence Lys-Val-Lys-Arg partially fits the Arg-Xaa-(Lys/Arg)-Arg consensus sequence for the proprotein convertase (PC) family of proteases (Nakayama 1997; Thomas 2002). To investigate the possibility that DREG is processed by a PC family protease, we utilized decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone, which is a competitive inhibitor of furin, a PC family protease. We examined the effect of this inhibitor on expression and processing of GFP-tagged hDREGa1-T841A in COS7 cells. Immunoblotting with anti-C antibody revealed a decrease in the amount of the ~140-kD fragment and appearance of a ~180-kD fragment. In contrast, processing of DREG was not inhibited when transfected cells were cultured in the presence of an MMP inhibitor, GM6001 (Fig. 9C). These results suggest that furin, or another member(s) of the PC family, is involved in the cleavage of the S2 site in DREG.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
A novel subtype B GPCR family, termed the LNB-TM7 family, has recently been described (Stacey et al. 2002). The LNB-TM7 family of proteins is characterized by the presence of a large extracellular domain, which is assumed to be involved in cell adhesion and signalling. Fredriksson et al. (2003) have recently searched the public human genome database using Hidden Markov Models based the structure of the previously known LNB-TM7, and found that there exist at least 30 human GPCRs of the LNB-TM7 type. In this study, we also searched the human genome database in the Sanger Center and identified a novel GPCR composed of a large extracellular domain containing a CUB domain, a PTX domain, an HBD, and a GPS domain. The CUB domain is characterized by four conserved Cys residues that form two disulphide bonds. Proteins containing the CUB domain have diverse functions, but most often are involved in development. The PTX domain is found in the Pentraxin family of proteins, which have been shown by electron microscopy to form complexes consisting of a discoid arrangement of five non-covalently bound subunits. The HBD is known to contain four conserved Cys residues, which probably form disulphide bridges. HBDs are present in many hormone-binding receptors, including most of the family B GPCRs and some of the LNB-TM7 family members. The functions of most of the LNB-TM7 receptor family members are unknown, although their tissue distributions suggest that many of them may have important functions in the nervous or immune systems. In addition to expression in the adult lung, DREG was expressed in the heart and the caudal somite during embryogenesis. Although the physiological function of DREG remains unknown, we speculate that DREG may play a role in cell differentiation, cell polarity and in the setting up of the segmental body plan. The TM7 domain and intracellular regions of DREG are very similar to those of HE6, another member of the LNB-TM7 family (Osterhoff et al. 1997). However, HE6 does not possess any other domains in its amino-terminus and its expression pattern is different from that of DREG. It is possible that DREG shares an intracellular signal transduction pathway with other LNB-TM7 family members, but has a different function specified by its extracellular region.

Like other members of the LNB-TM7 family, DREG possesses a Cys-rich proteolysis domain, called the GPS domain. Using fusion proteins consisting of the DREG extracellular domain and the Fc portion of human IgG1, we determined the cleavage site to be between residues Phe840 and Thr841. Our data agree well with the previously reported cleavage sites in CIRL, Ig-Hepta, and EMR4 (Krasnoperov et al. 1997; Abe et al. 2002; Stacey et al. 2002). Most of the point mutations we generated in the GPS domain rendered the protein resistant to cleavage, and resulted in its accumulation in intracellular vesicles (Figs 5 and 6). These results suggest that the entire GPS domain is important for the cleavage and proper trafficking of the receptors. It remains unknown why only the T841A mutant, which was also resistant to proteolytic processing at the GPS domain, could be transported to the cell membrane.

Amino-terminal sequence analyses revealed another cleavage site, termed S2, that is present in the middle of the extracellular domain of DREG. The amino acid sequence Lys-Val-Lys-Arg present just upstream of both the human and mouse S2 sites is a close match for the consensus recognition sequence of the PC family protease furin: Arg-Xaa-(Arg/Lys)-Arg (Nakayama 1997; Thomas 2002). In addition, DREG has a Lys at a position four amino acids upstream of the S2 site, and possesses a basic amino acid at a position six amino acids upstream of the S2 site (Lys in human and Arg in mouse). Thus, DREG is likely to be a substrate for furin. Consistent with this hypothesis, the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone was found to inhibit the processing of DREG at the S2 site in COS7 cells. This unprocessed form of DREG migrated more slowly in SDS-PAGE, similar to the migration of several of the GPS cleavage site mutants, except for T841A, suggesting that it may be subjected to post-translational modification, probably glycosylation.

The hypothetical maturation pathway of DREG is illustrated in Fig. 10. In the ER, pro-DREG may be processed by the signal peptidase and by a putative GPS protease. Although the GPS protease has not been identified, it has been reported that processing at the GPS domain occurs in the ER. DREG may be subsequently transported from the ER to the cell membranes through the Golgi apparatus, and is glycosylated during translocation. Because most of the PC family members are predominantly localized in the trans-Golgi network (Nakayama 1997; Thomas 2002), DREG may be processed at the S2 site intracellularly to produce an amino-terminal fragment. It is interesting to speculate that this fragment, containing the CUB and PTX domains, may require oligomerization for its function.



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  		Figure 10  A proposed model of DREG processing and function. DREG is synthesized as a large protein of over 1000 amino acids (for example 1221 amino acids in the case of hDREG{alpha}1). It is cleaved by a signal peptidase and a putative GPS protease in the ER, and cleaved again by a processing enzyme of the PC family. This processing releases the amino-terminal fragment as a putative ligand and enables it to signal on distant cells. A carboxy-terminal TM7 region is located in the plasma membrane, and acts as a receptor. A nature of the stalk region is unknown, but is supposed to be non-covalently associated with the TM7 regions, similar to other members of the LNB-TM7 family.

 
In summary, we have identified and characterized a novel LNB-TM7 family protein, DREG. The identification of two proteolytic sites in this protein raises the attractive possibility that DREG may have a dual role, functioning both as a receptor and a ligand. It would be interesting to investigate this possibility using soluble forms of recombinant DREG-Fc chimera proteins.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Materials

Chemicals and reagents were obtained from Sigma, Wako, Toyobo, and TAKARA unless otherwise specified. The silver staining kit (2D-silver stain II ‘DAIICHI’) and 2–15% polyacrylamide gels were from Daiichi Pure Chemicals Co. Ltd. Tokyo. Furin inhibitor I (decanoyl-Arg-Val-Lys-Arg-CMK) and matrix metalloproteinase (MMP) inhibitor GM6001 were purchased from CALBIOCHEM.

Isolation of full-length DREG cDNA clones

The human genomic sequence encoding a novel human GPCR was used to search the expressed sequence tag (EST) database for homologous sequences. An EST clone (GENBANK accession No. AA796299) that contained a 7TM region and 3' untranslated region (UTR) was found in a mouse cDNA library from the Incyte EST database. To amplify the 5' sequence, a plasmid cDNA library derived from mouse 17-day embryo (Clontech) was used as a template for PCR with the GAL4AD primer (Clontech) and the DREG-specific primer 5'-CACAAGAGCAATGTACATGTGG-3' (nucleotides 2891–2912 of the (–) strand of mDREG). A fragment of 1320 bp was obtained and sequenced, and this fragment was found to contain some but not all of the 5' region of DREG. A second amplification was performed with the same library using the GAL4AD primer and more 5' proximal DREG-specific primer 5'-CTGTGTCTCAATGTTCACATG-3' (nucleotides 2491–2511 of the (–) strand of mDREG), a fragment of 2060 bp was amplified. This fragment contained the predicted start site of the open reading frame and in-frame termination codon upstream of the initiation codon. To obtain the human cDNA clone, the 4.1 kb insert from the mouse EST clone was used to screen a human foetal brain cDNA library (Stratagene). Approximately 5 x 106 lambda phage clones were screened, and two positive clones that contained inserts of 5.1 kb and 5.0 kb, respectively, were isolated. To amplify the 5' sequence, a human keratinocyte MATCHMAKER cDNA library (Clontech) was used as a template for PCR using the GAL4AD primer and the DREG-specific primer 5'-TATTGCTTGGAAGACCAATGC-3' (nucleotides 2450–2451 of the (–) strand of hDREG{alpha}1). The final 5' end sequences of human and mouse cDNAs were derived from at least three independent experiments, and both strands of the isolated clones were sequenced with the BigDye terminator cycle sequencing kit on an DNA sequencer (Applied Biosystems).

RT-PCR

Two sets of mouse DREG-specific oligonucleotide primers (CUB +PTX domain sense primer, 5'-CCAATCCTTCCGGTACCTTTACGTC-3', and anti-sense primer, 5'-GGATCAGGTAGGAACCACAGCTCAG-3'; 3' UTR sense primer, 5'-GATGCTCACGGGTTCTGTTCCAGTG-3', and anti-sense primer, 5'-GAACCCTGAGCATGGGGCTAAAGC-3') were used to amplify 948-bp and 886-bp fragments of mDREG cDNA, respectively.

Whole-mount in situ-hybridization

A 2.6 kb cDNA fragment encoding the extracellular region of mouse DREG was amplified by PCR and cloned into pCRII (Invitrogen) in two orientations. A second probe to detect a different region of DREG was created from the EST clone AA796299 [GenBank] , which contained an insert of 4.1 kb fragment, and which was linearized by XhoI or NotI. Sense and anti-sense probes were synthesized using T7 or SP6 RNA polymerase using the DIG RNA labelling kit (Roche Molecular Biochemicals). Embryos were dissected in phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 2 h at 4 °C. The embryos were washed two times with PBS containing 0.1% Tween 20 (PBS-T), dehydrated in 100% methanol and stored at –20 °C. The embryos were subsequently rehydrated through a methanol-PBS-T series and washed two times in PBS-T. Hybridization was carried out with an InsituPro (ABIMED AnalysenTechnik GmbH, Deuschland) machine according to the manufacturer's instructions. The colour reaction was initiated by washing the embryos in NTMT (100 mM NaCl, 100 mM Tris-Cl, pH 9.5, 50 mM MgCl2, 0.1% Tween 20) containing 4.5 µL/mL NBT (75 mg/mL nitroblue tetrazolium salt) and 3.5 µL/mL BCIP (50 mg/mL 5-bromo-4-chloro-3-indolyl phosphate toluidine). When staining was satisfactory, the embryos were washed three times with PBS-T.

Plasmids

The open reading frame of DREG was amplified by PCR and subcloned into the mammalian expression vector pcDNA3.1(+), pcDNAmycHis(A) (Invitrogen), and pEGFP-N1 (Clontech). Mutants of DREG were generated by PCR-based mutagenesis. The authenticity of all mutants was verified by DNA sequencing. For the construction of vectors expressing human Fc fusion proteins, the Fc region was amplified by PCR using pME18S-mCRTAM-hIg (gift from S. Kimura) as a template and subcloned into pcDNA3.1(+). DNA fragments encoding the wild-type and mutant DREG extracellular domains were generated by PCR and subcloned immediately upstream of the Fc region in pcDNA3.1(+).

Antibodies

For anti-DREG antibody production, DNA fragments encoding the CUB domain of mouse DREG (residues 41–149) and the carboxy-terminal cytosolic domain of human DREG{alpha}1 (residues 1117–1187) were amplified by PCR and cloned into pET16b (Novagen). The constructs were transformed into E. coli BL21 (DE3) and used for fusion protein production. Fusion proteins were found to be insoluble and therefore purified under denaturing conditions on a TALON metal affinity resin (Clontech) column. Anti-DREG polyclonal antibodies were raised in rabbits by immunizing them with the recombinant proteins. Antibodies were purified by affinity chromatography using columns to which the antigens used for immunization had been linked.

Cell cultures and transient transfection

COS7 and HEK293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum. Transfection into cells was done by the FuGENE 6 method according to the manufacturer's protocols (Roche). For preparation of total cell extracts, cells were washed twice by PBS and fixed by ice-cold trichloroacetic acid for 30 min. The cell pellets were suspended and sonicated in 9 M urea containing 2% TritonX-100 and 1% dithiothreitol, and then a one-fourth volume of 10% LiDS was added to cell lysates. They were neutralized by 1 M Tris and used for immunoblotting.

Purification of DREG-Fc

HEK293T cells were transfected with an expression vector encoding the DREG-Fc fusion protein. The culture medium was replaced with serum-free Opti-MEM I (Invitrogen) 15 h post transfection and incubated for a further 96 h. Conditioned medium was collected, centrifuged, passed through a 0.22-µm filter membrane and loaded on to protein A-Sepharose 4 Fast Flow (Amersham Biosciences) column. Protein elution was performed according to the manufacturer's protocols. For amino-terminal sequencing, purified DREG-Fc fusion proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The protein band of interest was excised from the membrane, and amino-terminal sequence was determined by automated Edman degradation using Procise-cLc (Applied Biosystems).

Cell staining

COS7 cells were cultured on glass coverslips, and transiently transfected with DREG expression plasmids. Twenty-four h after transfection, cells on coverslips were fixed with 3.7% formaldehyde in PBS for 10 min at 37 °C, and treated with 0.5% Triton X-100 in PBS for 10 min. After blocking with PBS containing 3% bovine serum albumin and 1% foetal bovine serum, the coverslips were incubated with the anti-GFP antibody (anti-AFP mAb 3E6, Qbiogene Inc.) and then washed three times with PBS. Reacted proteins were detected by FITC-conjugated goat anti-mouse secondary antibody (ICN Biomedicals). All fluorescence images were obtained with a confocal laser microscope LSM510 (Carl Zeiss).


   Acknowledgements
 
We thank S. Aihara and T. Matsuda for their technical assistances; Y. Yoshimura for peptide sequencing; S. Kimura for providing the mCRTAM-hIgG expression plasmid. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Uehara Memorial Foundation, Tokyo.


   Footnotes
 
Communicated by: Tadashi Yamamoto

aPresent address: Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, and Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Back

* Correspondence: E-mail: akiyama{at}iam.u-tokyo.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Abe, J., Fukuzawa, T. & Hirose, S. (2002) Cleavage of Ig-Hepta at a ‘SEA’ module and at a conserved G protein-coupled receptor proteolytic site. J. Biol. Chem. 277, 23391–23398.[Abstract/Free Full Text]

Ballesteros, J. & Palczewski, K. (2001) G protein-coupled receptor drug discovery: implications from the crystal structure of rhodopsin. Curr. Opin. Drug. Discov. Devel. 4, 561–574.[Medline]

Bockaert, J. & Pin, J.P. (1999) Molecular tinkering of G protein-coupled receptors: an evolutionary success. EMBO J. 18, 1723–1729.[CrossRef][Medline]

Curtin, J.A., Quint, E., Tsipouri, V., et al. (2003) Mutation of Celsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse. Curr. Biol. 13, 1129–1133.[CrossRef][Medline]

Fredriksson, R., Gloriam, D.E., Hoglund, P.J., Lagerstrom, M.C. & Schioth, H.B. (2003) There exist at least 30 human G-protein-coupled receptors with long Ser/Thr-rich N-termini. Biochem. Biophys. Res. Commun. 301, 725–734.[CrossRef][Medline]

Fredriksson, R., Lagerstrom, M.C., Hoglund, P.J. & Schioth, H.B. (2002) Novel human G protein-coupled receptors with long N-terminals containing GPS domains and Ser/Thr-rich regions. FEBS. Lett. 531, 407–414.[CrossRef][Medline]

George, S.R., O'Dowd, B.F. & Lee, S.P. (2002) G-protein-coupled receptor oligomerization and its potential for drug discovery. Nature Rev. Drug. Discov. 1, 808–820.[CrossRef][Medline]

Gray, J.X., Haino, M., Roth, M.J., et al. (1996) CD97 is a processed, seven-transmembrane, heterodimeric receptor associated with inflammation. J. Immunol. 157, 5438–5447.[Abstract]

Ichtchenko, K., Bittner, M.A., Krasnoperov, V., et al. (1999) A novel ubiquitously expressed alpha-latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors. J. Biol. Chem. 274, 5491–5498.[Abstract/Free Full Text]

Krasnoperov, V.G., Bittner, M.A., Beavis, R., et al. (1997) alpha-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor. Neuron 18, 925–937.[CrossRef][Medline]

Krasnoperov, V., Lu, Y., Buryanovsky, L., et al. (2002) Post-translational proteolytic processing of the calcium-independent receptor of alpha-latrotoxin (CIRL), a natural chimera of the cell adhesion protein and the G protein-coupled receptor. Role of the G protein-coupled receptor proteolysis site (GPS) motif. J. Biol. Chem. 277, 46518–46526.[Abstract/Free Full Text]

McMillan, D.R., Kayes-Wandover, K.M., Richardson, J.A. & White, P.C. (2002) 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. 277, 785–792.[Abstract/Free Full Text]

Nakayama, K. (1997) Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem. J. 327, 625–635.

Nechiporuk, T., Urness, L.D. & Keating, M.T. (2001) ETL, a novel seven-transmembrane receptor that is developmentally regulated in the heart. ETL is a member of the secretin family and belongs to the epidermal growth factor-seven-transmembrane subfamily. J. Biol. Chem. 276, 4150–4157.[Abstract/Free Full Text]

Obermann, H., Samalecos, A., Osterhoff, C., et al. (2003) HE6, a two-subunit heptahelical receptor associated with apical membranes of efferent and epididymal duct epithelia. Mol. Reprod. Dev. 64, 13–26.[CrossRef][Medline]

Osterhoff, C., Ivell, R. & Kirchhoff, C. (1997) Cloning of a human epididymis-specific mRNA, HE6, encoding a novel member of the seven transmembrane-domain receptor superfamily. DNA Cell. Biol. 16, 379–389.[Medline]

Ponting, C.P., Hofmann, K. & Bork, P. (1999) A latrophilin/ CL-1-like GPS domain in polycystin-1. Curr. Biol. 9, R585–R588.[CrossRef][Medline]

Skradski, S.L., Clark, A.M., Jiang, H., et al. (2001) A novel gene causing a mendelian audiogenic mouse epilepsy. Neuron 31, 537–544.[CrossRef][Medline]

Stacey, M., Chang, G.W., Sanos, S.L., et al. (2002) EMR4, a novel epidermal growth factor (EGF)-TM7 molecule up-regulated in activated mouse macrophages, binds to a putative cellular ligand on B lymphoma cell line A20. J. Biol. Chem. 277, 29283–29293.[Abstract/Free Full Text]

Stacey, M., Lin, H.H., Gordon, S. & McKnight, A.J. (2000) LNB-TM7, a group of seven-transmembrane proteins related to family-B G-protein-coupled receptors. Trends Biochem. Sci. 25, 284–289.[CrossRef][Medline]

Thomas, G. (2002) Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nature Rev. Mol. Cell. Biol. 3, 753–766.[CrossRef][Medline]

Usui, T., Shima, Y., Shimada, Y., et al. (1999) Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity under the control of Frizzled. Cell 98, 585–595.[CrossRef][Medline]

Received: 22 December 2003
Accepted: 15 March 2004





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