Type IV collagen is an activating ligand for the adhesion G protein-coupled receptor GPR126

Gpr126 binds specifically to type IV collagen

To identify ligands for Gpr126, we expressed a secreted, Fc-tagged protein containing the N-terminal fragment of zebrafish Gpr126 (Gpr126-NT-Fc, Fig. 1A) in human embryonic kidney 293 (HEK293) cells. Previous studies show that Gpr126 and type IV collagen in the extracellular matrix are involved in myelination (26, 34, 35), and a recent paper identified type III collagen as an activating ligand for Gpr56 (22). To search for specific interactions between Gpr126 and different collagen proteins, we performed a series of pulldown assays with the Gpr126 N-terminal Fc fusion protein and purified collagen proteins, types I, II, III, IV, and V. Biotinylated collagen proteins were incubated with streptavidin-agarose and conditioned media from transfected HEK293 cells expressing Gpr126-NT-Fc. Bound proteins were analyzed by immunoblot with an antibody specific to Fc to determine if any collagen proteins interacted with the Gpr126 N-terminal region. Gpr126-NT-Fc robustly interacted with type IV collagen, but not the other four collagen types (Fig. 1B). A control protein representing only the Fc tag of the fusion protein did not bind to any of the collagen proteins or to BSA (fig. S1). In reciprocal experiments, biotinylated collagen proteins and media containing Gpr126 NT-Fc were incubated with Protein A-agarose. Again, Gpr126-NT-Fc interacted robustly with type IV collagen, but not with the other four collagen types or BSA (Fig. 1D). These results demonstrate that the N-terminal region of Gpr126 specifically binds to type IV collagen.

Type IV collagen activates Gpr126 signaling

Previous genetic and biochemical studies show that Gpr126 signals through Gα_s to stimulate cAMP in vivo and in heterologous cell systems (26-29). In addition, forskolin promotes myelination in cultured Schwann cells (36), and Schwann cells express gpr126 and secrete type IV collagen prior to the onset of myelination (26, 37). In combination, our results and these previous studies suggested that type IV collagen may be an autocrine signal that increases cAMP concentration in Schwann cells. To investigate this possibility, we treated Schwann cells purified from rat sciatic nerve with type IV collagen or PBS for 1 hour. Treatment with type IV collagen significantly increased cAMP concentrations in Schwann cells (Fig. 2A), supporting the possibility that type IV collagen is an endogenous autocrine inducer of cAMP signaling in Schwann cells.

To further analyze the interaction between type IV collagen and Gpr126, we used a heterologous overexpression system. HEK293 cells were transfected with a full-length Gpr126 expression vector or a control plasmid and treated with PBS or type IV collagen. Treatment with type IV collagen significantly increased cAMP concentrations in cells transfected with Gpr126, but had no effect on control cells lacking the receptor (Fig. 2B). Transfection with Gpr126 caused a smaller but significant increase in cAMP concentration in the absence of collagen treatment (Fig. 2B). Ligand-independent signaling has been commonly observed in similar experiments with other GPCRs, because overexpression increases the population of receptors in the active conformation even in the absence of ligand (38).

To test the temporal response of Gpr126-expressing cells to collagen IV, we performed a time course study. In response to 3 μg/mL of type IV collagen, cAMP concentration increased in Gpr126-expressing HEK293 cells as early as 10 min, reached peak concentrations after 60 min, and declined after 120 min (Fig. 2C). The reduction at later time points could be caused by internalization of the receptor after ligand binding, similar to the aGPCR CD97 and its ligand CD55 (18). There is also evidence that Gpr126 couples to Gα_i (29), and it is possible that extended activation of the receptor leads to inhibition of cAMP by coupling to different effectors.

In a dose-response analysis, we treated Gpr126-expressing HEK293 cells with a range of type IV collagen concentrations and measured cAMP concentrations after 1 hour. Gpr126-expressing cells increased cAMP production in response to concentrations of type IV collagen as low as 0.06 μg/mL and produced near-maximal amounts of cAMP at concentrations above 0.6 μg/mL (Fig. 2 D). Analysis of the data determined that the concentration that induced a half-maximal response (EC₅₀) was 0.2 μg/mL, corresponding to approximately 0.7 nM.

To explore the possibility that other collagen proteins activate Gpr126 signaling, we treated HEK293 cells transfected with the Gpr126 expression plasmid or a control vector with PBS or 3 μg/mL collagen type I, II, III, IV, or V for 1 hour. As shown above, type IV collagen robustly increased cAMP concentration in Gpr126-expressing cells, but the other four collagen proteins had no effect (Fig. 2E). In summary, these results indicate that type IV collagen specifically binds the N-terminal region of Gpr126, and that this interaction activates the receptor, leading to an increase in the second messenger cAMP.

The CUB and Pentraxin domains of Gpr126 bind to type IV collagen

The N-terminal region of Gpr126 contains several conserved elements, including the CUB, PTX, hormone binding, and GAIN domains (39, 40). We focused on the CUB and PTX domains because these domains in other proteins can bind to various collagen-like proteins, including type IV collagen (41-44). We analyzed the binding activity of two complementary Fc fusion proteins (Fig. 3A). CUBPTX-Fc contained a signal peptide, the CUB domain, and the PTX domain followed by an Fc tag (amino acids 41-336 of Gpr126). The complementary protein, ΔCUBΔPTX-Fc, contained a signal peptide, the rest of the Gpr126 N-terminal region, and an Fc tag (amino acids 337-827 of Gpr126). The fusion proteins were harvested from conditioned media of transfected HEK293 cells and analyzed in pulldown experiments with biotinylated collagen type IV and streptavidin-agarose. Both Gpr126NT-Fc and CUBPTX-Fc strongly bound collagen type IV, in contrast to ΔCUBΔPTX-Fc, which displayed no detectable interaction (Fig 3B).

To quantitatively compare the affinities of Gpr126NT-Fc and CUBPTX-Fc for type IV collagen, we used surface plasmon resonance binding assays. Collagen type IV was coupled to the surface of the sensor chip, and the affinity-purified Gpr126-NT-Fc protein was introduced at various concentrations, with the surface being regenerated between trials. Gpr126NT-Fc bound to collagen IV with high affinity in duplicate trials (Fig. 3C). The mean calculated K_D value was 1.3 nM, similar to the EC₅₀ for activation (Fig. 2D), with association and dissociation constants of 3.06 ± 0.05 × 10⁵ (1/Ms) and 4.04 ± 0.8 × 10^-4 (1/s), respectively. In similar experiments, CUBPTX-Fc also bound to collagen IV with high affinity in duplicate trials (Fig. 3D). The mean calculated K_D value was 1.2 nM, with association and dissociation constants of 2.78 ± 0.4 × 10⁶ (1/Ms) and 3.33 ± 1 × 10^-3 (1/s), respectively. These results indicate that the region of Gpr126 containing the CUB and PTX domains binds type IV collagen with high affinity and specificity.

The N-terminal region of Gpr126 controls its signaling activity

Functional studies of aGPCRs have led to different models about the role of the N-terminal region in receptor activation (12-17, 45). One model is that the cleaved N-terminal region antagonizes receptor signaling activity, so that truncation of the N-terminus can create a constitutively active receptor (12-16). To test whether this scenario might apply to Gpr126, we created FLAG-tagged Gpr126 constructs representing the full-length and N-terminally cleaved receptor (Fig. 4A). Both constructs were expressed at similar amounts, as indicated by immunostaining of transfected cells with an antibody against FLAG (fig. S2). In lysates of transfected cells (Fig. 4B, right lanes), analysis of Gpr126-FLAG detected multiple bands, including a 35 kDa protein corresponding to the C-terminal region after GPS cleavage (bottom arrow), and higher molecular weight species representing modified and uncleaved forms of Gpr126. Multiple proteins were also detected in samples from cells transfected with Gpr126ΔNT-FLAG (Fig. 4B), including the 35 kDa protein and an abundant species with an apparent molecular weight of approximately 60 kDa (top arrow). The 60 kDa protein could be a ubiquitinated form, which is observed in the active species of other GPCRs, including the adhesion GPCR GPR56 (12, 46, 47). We also determined the extent of surface expression for each construct using a standard surface biotinylation assay, in which surface proteins were biotinylated and then pulled down with streptavidin beads (Fig. 4B, left lanes). The 35kDa protein produced from cells transfected with Gpr126-FLAG and Gpr126ΔNT-FLAG was expressed at the surface at similar amounts, consistent with similar experiments on GPR56 showing that the cleaved form of the receptor predominates at the cell surface (12). In control analyses of the same lysates, we detected no surface-biotinylated actin protein (Fig. 4B lower panel).

We next examined the activity of the Gpr126ΔNT-FLAG receptor and its ability to respond to type IV collagen. As in the experiments described above, treatment with type IV collagen increased cAMP concentrations in HEK293 cells expressing the full-length, FLAG-tagged receptor (Fig. 4C), whereas cells transfected with a control vector showed no response to type IV collagen. Cells expressing Gpr126ΔNT-FLAG showed a markedly high concentration of cAMP (Fig. 4C), which did not increase further after addition of type IV collagen (Fig. 4C). These experiments provide evidence that the N-terminal region of Gpr126 inhibits signaling in absence of ligand.

The gpr126^st86 mutation truncates Gpr126 after the CUB and PTX domains and disrupts myelination and inner ear development, but not the heart

In a genetic screen for mutations that disrupt myelinated axons, we recovered a new allele of gpr126, gpr126^st86, that introduces a premature stop codon at position 406 and truncates the protein between the pentraxin and hormone binding domains (Fig. 5, A and B). The amount of gpr126 mRNA was similar in wild-type and gpr126^st86/st86 homozygotes, and therefore it is possible that the mutant allele produced a truncated protein containing the CUB and PTX domains (fig. S3). Like other strong mutant alleles of gpr126, homozygous gpr126^st86/st86 mutants lack expression of mbp encoding myelin basic protein, in Schwann cells and have swollen ears (Fig. 5, C and D). Heart development appeared to be normal in gpr126^st86/st86 mutants (Fig. 5D). Like gpr126^st49/st49 mutants (26, 28), homozygous gpr126^st86/st86 mutants survived to adulthood, further confirming that the mutation did not cause lethal heart abnormalities and allowing us to examine the possible maternal functions of gpr126. To examine the phenotype caused by loss of maternal (M) and zygotic (Z) function, we crossed a heterozygous gpr126^st86/+ male to a gpr126^st86/st86 homozygous mutant female. The resulting Mgpr126^st86/+ mutants had a wild-type phenotype, and MZgpr126^st86/st86 mutants resembled Zgpr126^st86/st86 mutants, having swollen ears and normal hearts (Fig. 5D). If the previous proposal is correct that a region of Gpr126 has a function in heart development in zebrafish (32), our analysis suggests that this function is fulfilled by a protein fragment containing the first 405 amino acids of Gpr126—the region encoded 5′ to the premature stop codon in the gpr126^st86 mutation.

Constructs

The Gpr126NT-Fc was cloned into pFUSE-hIgG2-Fc2 vector (Invivogen) using homologous recombination method with the GenScript CloneEZ kit per manufacturer's protocol. Target DNA was amplified by PCR (primers: forward 5′-GTCACGAATTCGATATCGGCCATGGTTCAGAGCTGCCAGAGTAGCACC-3′ and reverse, 5′-TGGGCAAGGTGGGCACTCCACAGATCTGAGGTGATTACACAAGCAGAC-3′) and inserted into BglII linearized vector using CloneEZ kit. The same method was used for CUBPTX-Fc (primers: forward, 5′- CTTGCACTTGTCACGAATTCGTGCAATGTGGTGCTCACCGAC-3′ and reverse, 5′- TGGGCAAGGTGGGCACTCCACATCCCAGTCAACGACATTACC-3′) and ΔCUBΔPTX-Fc (primers: forward, 5′-CTTGCACTTGTCACGAATTCGCATTCCTACTGGACTATCCCA-3′ and reverse, 5′-TGGGCAAGGTGGGCACTCCACGAGGTGATTACACAAGCAGAC-3′). C-terminal FLAG tag Gpr126 was cloned into p3XFLAG-CMV-14 vector (Sigma Aldrich) using standard cloning techniques and restriction sites HindIII and ClaI (primers: forward, 5′-AAATTTAAGCTTATGATTTCGTTCGTTCATCAGTGGTCGG-3′ and reverse, 5′-AAATTTATCGATAATTGCAGGGTACTATCTGCATTACT-3′). To create the Gpr126ΔNT construct, the signal peptide was first cloned into p3XFLAG-CMV-14 vector using HindIII and ClaI restriction sites (primers: forward, 5′-AAATTTAAGCTTATGATTTCGTTCATCAGTGGTCGG -3′ and reverse, 5′-AAATTTATCGATGACAACGCGGTCATAGATGCAGCC-3′). The Gpr126ΔNT fragment was amplified (primers: forward, 5′-ATCTGCCTTTCCACCTCAGTGGCAACACACTTTGGCATCCTAATGGAT-3′ and reverse, 5′-TGGTACCGATATCAGATCTATCGATGATTGCAGGGTACTATCTGCATT-3′) and inserted into the signal peptide containing p3XFLAG-CMV-14 vector at the XhoI site using the Clone EZ kit. A recent paper (30) noted that the reference sequence for the zebrafish gpr126 gene codes for Cys⁸⁰⁴, but that the wild-type zebrafish strains they analyzed encoded Trp at that position. Our analysis also showed that wild-type fish strains code for Trp at this position, and our constructs all contained Trp at this position. The sequences of all constructs were verified.

Purification of Fusion proteins

HEK293 cells were transfected with empty pFUSE vector or expression vectors for our different Gpr126-Fc fusion proteins using Lipofectamine 2000 following the manufacturer's standard protocol. Cells were incubated at 37 °C for 3 days. The conditioned media was collected and concentrated using Amicon Ultra 15 centrifugation filters. Concentrated conditioned media was then incubated overnight at 4°C with Protein A agarose to purify fusion proteins. Proteins were then eluted from beads with 1 M NaCl or 0.1M glycine buffers and dialyzed into HEPES buffered saline [10 mM HEPES and 150 mM NaCl, pH 7.5]. Protein purity was determined by SDS-PAGE followed by silver stain, and the concentration determined by Bradford assay.

Pull down assays

Purified collagen proteins were obtained from Sigma Aldrich and were biotinylated using the Pierce EZ-link biotinylation kit. Equal quantities by weight of biotinylated collagen and Gpr126 NT-Fc were mixed and incubated overnight with streptavidin or Protein A agarose. Beads were washed in HEPES buffered saline [10 mM HEPES and 150 mM NaCl, pH 7.5] then eluted by addition of Laemmli sample buffer. Samples were resolved by SDS-PAGE followed by transfer to nitrocellulose membranes. The membranes were incubated in blocking buffer (2% nonfat dry milk, [50 mm NaCl, 20 mm HEPES, and 0.1% Tween 20] for 30 min and then incubated with primary antibody for 1 hour at room temperature. In cases where the primary antibody was not directly coupled to HRP, membranes were washed in blocking buffer and incubated with HRP-conjugated secondary antibody (GE healthcare) for 30 min. After washing to remove HRP-coupled antibodies, blots were visualized via ECL reagent (Pierce) followed by exposure to film.

cAMP measurements

HEK293 cells were transfected by standard protocol using Lipofectamine 2000 with empty vector or expression plasmid containing various Gpr126 constructs. Cells were treated with collagen for 1 hour unless otherwise indicated. The cells were lysed in 0.1 M HCl and, the intracellular cAMP concentration measured with an ELISA-based kit (EMD Millipore). To determine the EC₅₀, the dose-response data were analyzed with a four-parameter Hill equation using GraphPad Prism software.

Surface Plasmon Resonance (SPR)-based Biosensor Analysis

The interaction of Gpr126 variants with type IV collagen was analyzed by SPR using a BIACORE 3000 biosensor system (GE Healthcare). Protein dilutions were performed in HBS-N (10mM HEPES at pH 7.4, 150 mM NaCl) running buffer. Type IV collagen was immobilized onto CM5 biosensor chip by amine coupling chemistry using N-hydroxysuccinimide (NHS) and N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). To investigate binding of Gpr126NT-Fc or CUBPTX-Fc, proteins were diluted in HBS-N buffer and injected over the Type IV collagen surface. The experiments were performed at 25 °C using a flow rate of 30 μl/min. For each experiment, at least 5 different concentrations of Gpr126NT-Fc or CUBPTX-Fc were injected over each experimental and control flow cell. Dissociation was allowed to occur at the same flow rate for 300 sec, followed by running buffer alone for 2 min. The surface was regenerated with 0.5 N NaCl for 30 sec, followed by 1.0M NaCl using a flow rate of 100 μl/min, followed by running buffer alone for 2 min to enable stabilization of the baseline. Biosensor experiments were repeated a minimum of two times. All data were corrected for nonspecific binding by subtracting the signal measured in a control cell lacking immobilized ligand. Both data processing and kinetic fitting were performed using Scrubber software, version 2 (BioLogic Software, Pty., Australia) or BIAevaluation software 4.1 (Biacore).

Purification and culture of neonatal rat Schwann cells

Rat Schwann cells were acutely isolated from neonatal (P2) rat sciatic nerves by immunopanning, using the method of Cheng and Mudge (66). Briefly, isolated sciatic nerves were digested with collagenase/dispase and then incubated on sequential negative panning plates containing antibodies against CD45 (to deplete macrophages) and Thy 1.1 (to deplete fibroblasts). Schwann cells were then purified to about 99% purity by adherence to an O4 (anti-sulfatide) plate, and removed by trypsin. For cAMP assays, acute Schwann cells were plated on poly-D-lysine coated coverslips in defined, serum-free media (DMEM/SATO containing insulin, sodium pyruvate, L-glutamine, Thyroxine, B27, and N-acetyl-cysteine) in the absence of forskolin or growth factors. Cells were grown overnight prior to testing.

Fish strains and Genotyping

Zebrafish embryos were raised at 28.5°C and staged as described (67). The gpr126^st86 mutation was identified in an ENU-based screen for mutations that disrupted mbp mRNA expression, following previously described methods for mutagenesis and screening (25). The gpr126^st86 mutation failed to complement the previously described gpr126^st49 allele (26), and sequencing the gpr126 gene from mutants and wildtype siblings identified the lesion. To genotype individual animals in the phenotypic analyses, the gpr126^st86 mutation was scored by a PCR test of genomic DNA samples. We used primers to amplify genomic DNA spanning the lesion (5′-TGTAAGACTTTGGGTTGATTTGG-3′ and 5′-CATTGAGATATTATCGCCGTAGC-3′), and digested the products with the restriction enzyme DpnII. The gpr126^st86 mutation disrupts one of the two DpnII sites in the wild-type sequence.

Surface Biotinylation Assay

HEK293 cells transfected with Gpr126-FLAG or Gpr126ΔNT-FLAG were washed with PBS and suspended in PBS containing 2mM Sulfo-NHS-Biotin reagent (Pierce). The biotinylation reaction was incubated for 2 hours on ice. Cells were gently washed in 100 mM glycine solution to quench the reaction and then lysed in buffer with 1% Triton X-100, 300 mM NaCl, and 50 mM Tris. The lysate was cleared by high-speed centrifugation. After a portion was set aside to analyze total cell lysates, the remainder of the lysate was incubated with streptavidin agarose overnight at 4 degrees to pull down biotinylated proteins. Beads were washed, suspended in sample buffer [5 M Urea, 1% SDS, 10% glycerol, 5% β-mercaptoethanol, 50 mM Tris], and then resolved by SDS-PAGE followed by transfer to nitrocellulose membranes. Gpr126-FLAG surface expression was assessed by Western blot analysis using a FLAG antibody. As a control cytoplasmic protein, we analyzed the samples in immunoblots with an actin antibody (Thermo Scientific, MS-1295).

Analysis of FLAG constructs

For immunoblots, transfected HEK293 cells transfected with Gpr126-FLAG or Gpr126ΔNT-FLAG were lysed directly in a Urea based sample buffer [5 M Urea, 1% SDS, 10% glycerol, 5% β- mercaptoethanol, 50 mM Tris] and then sonicated on ice. For microscopy, HEK293 cells were transfected with empty vector, Gpr126-FLAG, or Gpr126ΔNT-FLAG, plated onto coverslips, and allowed to attach overnight. Cells were then fixed with 4% paraformaldehyde, washed, and incubated with an antibody against FLAG (1:2000) overnight at 4 °C. Cells were washed extensively and then incubated with Alexa Fluor goat anti-mouse 488 secondary (1:500) for 1 hour at room temperature. Slides were viewed using the 40× objective of a Zeiss LSM 5 Pascal confocal microscope and images were acquired with a constant setting for comparison across conditions using Zeiss LSM software. Mean gray value was used to compare expression of different FLAG-tagged constructs in transfected cells, measured using Image J software; FLAG-positive cells were traced and the mean gray value and area determined by the software. To determine transfection efficiency, cells were visualized with a Zeiss Axio Imager M2 microscope; DAPI stained nuclei were counted to determine the total number of cells, and transfected cells counted by visualizing FLAG antibody staining.

qRT-PCR Analysis

Total RNA was extracted from individual embryos using the RNeasy Micro Kit (Qiagen). Embryos were sorted according to the ear phenotypes prior to lysis and confirmed using a PCR-restriction digest assay as described above. cDNA was made using random hexamers and SuperScript III Reverse Transcriptase (Invitrogen). qPCR was performed on the Bio-Rad CFX384 Real-Time PCR Detection System with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). gpr126 transcripts were detected using the forward primer 5′-TGTCGTTGACTGGGATCATT-3′ and reverse primer 5′-CGGTTCCTGGTGAAAGAGTT-3′. Values were normalized to the expression of ef1α.

Statistical Analysis

Single comparisons for statistical significance were done using an unpaired t-test with GraphPad Prism software. Statistical significance was deemed P<0.05.