All chemicals, unless stated otherwise were purchased from Sigma-Aldrich (St. Louis, MO, United States).

Online server SWISS-MODEL (http://swissmodel.expasy.org/) was used to generate homology based models of mouse RBPR2, mouse STRA6, and zebrafish Stra6. The model with maximum coverage and lowest Z score for each was selected for further studies. The template selected (by online server SWISS-MODEL) was the cryoEM structure of STRA6, receptor for retinol (PDB ID 5sy1, Chain B) which showed nearly 44% sequence identity and Q mean close to −5 for STRA6 identity; and nearly 22% sequence identity and Q mean close to −7 for RBPR2. The structure for RBP4 was obtained from PDB database (RSCB PDB ID: 2wqa, Chain E). The models generated were used for docking studies to analyze the protein-protein interactions employing the online data-driven docking program HADDOCK. HADDOCK requires a set of ambiguous interaction restraints (AIRs) at the binding interface that are divided into “active” and “passive” categories where active residues are those directly implicated in binding from experimental data and passive residues are their near neighbors. The docking process included a rigid body energy minimization step. The residues S294, Y295 and L296 for mouse RBPR2 were assigned as active residues for interaction as per the previous published papers to be essential for binding (Alapatt et al., 2013; Chen et al., 2016). The residues between 8 and 12 Å from these three residues were defined as passive. HADDOCK clustered 187 structures in 11 cluster (s), which represents 93.5% of the water-refined models HADDOCK generated for RBP4-Stra6. HADDOCK clustered 138 structures in 12 cluster(s), which represents 69.0% of the water-refined models HADDOCK generated for RBP4-RBPR22. The top cluster with the minimal haddock scores of −95.1 +/− 2.0; and lowest Z-score of −1.5 for RBP4-RBPR2 was selected for analysis (van Zundert et al., 2016; Waterhouse et al., 2018).

Total RNA (∼2 μg) from liver of a 2-month-old wild-type C57/B6 mouse was reverse transcribed using the SuperScript One-Step RT-PCR for LongTemplates system (Invitrogen, Grand Island, NY). The full-length mouse Rbpr2 cDNA was amplified by using mouse gene specific Rbpr2 primers with the Expand High Fidelity PCR system (Roche, Indianapolis, IN, USA). The amplified Rbpr2 cDNA product was cloned in frame into the pCDNA3.1 V5/His TOPO vector (Invitrogen, Carlsbad, CA). Appropriate construction of the wild-type Rbpr2 plasmid in the pCDNA 3.1 V5/His TOPO vector (pRbpr2-V5) was verified by sequence analysis of both strands (GENEWIZ, USA) and by comparing the sequences to the reference mouse Rbpr2/Stra6like cDNA sequences deposited in Ensembl (www.ensembl.org). The WT-Rbpr2 plasmid was used as a template and mutagenic Rbpr2 primer pairs were used to engineer each of the RBP4 binding residue mutants by in vitro site-directed mutagenesis (Quick Change II XL: Stratagene/Agilent, Santa Clara, CA), as previously achieved (Shi et al., 2017; Solanki et al., 2020). Appropriate construction of the WT-Rbpr2 and mutant-Rbpr2 plasmids were verified by DNA sequence analysis of both strands using pCDNA3.1 vector primers (GENEWIZ, USA).

Mouse NIH3T3 cells obtained from American Type Tissue Culture (ATCC-1658) were maintained in high-glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin sulfate and cultured at 37°C with 5% CO₂. NIH3T3 cells were used in this experiment as they are a well-established cell line to study the in vitro function of vitamin A membrane receptors for RBP4 binding and ROL transport (Amengual et al., 2014; Shi et al., 2017; Lobo et al., 2018; Solanki et al., 2020). To generate constitutively expressing mouse RBPR2 in NIH3T3 cells, parental NIH3T3 or NIH3T3/LRAT expressing cells were transiently transfected with the pRbpr2-V5 plasmid, as described previously (Solanki et al., 2020). Approximately 40 h post transfection, media was replaced to contain 400 μg/mL Geneticin (G418) selection agent. After 2 weeks of selection with G418, surviving individual cells (n = 12) were selected by placing cloning rings around each surviving cell. Each clonal cell was then carefully detached by adding 10 μL of trypsin into each clonal ring. Detached cells were transferred to 6-well culture plates containing 200 μg/mL G418 selection media. Once individual clones reached ∼80% confluence they were expanded into 100 mm dishes containing 200 μg/mL of G418 selection media. To confirm stable integration of the Rbpr2 gene and expression in these cells, we isolated total protein from each clone and subject them to western blot analysis. By using the V5-primary antibody, we detected the V5-tagged RBPR2 protein.

Cell lines were grown on coverslips and fixed in a freshly prepared mixture of 4% formaldehyde in 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, and 2 mM potassium phosphate monobasic, pH 7.4) for 30 min at room temperature and processed as previously described (Lobo et al., 2010; Lobo et al., 2013; Solanki et al., 2020; Solanki et al., 2021). Parental NIH3T3 cells were transiently transfected with the pRbpr2-V5 plasmid, as described previously (Solanki et al., 2020). Subcellular localization of the recombinant mouse Rbpr2-V5 in NIH3T3 cells was achieved by exposure to the anti-V5 primary antibody (which detects the V5-tagged RBPR2) followed by the anti-rabbit conjugated Alexa 488 secondary antibody staining (Invitrogen, Carlsbad, CA). Cells were examined under a Zeiss LSM 510 UV Meta confocal microscope with an HCX Plan × 40 numerical aperture 1.4 oil immersion objective lens (Zeiss, Jena, Germany). Images were acquired with the Zeiss confocal software, version 2.0. All experiments were carried out in triplicate. Approximately 55–75 cells from 7–9 fields were imaged/counted per experiment (Lobo et al., 2010; Lobo et al., 2013; Rohrer et al., 2021).

RBP4 cDNA cloned into the pET3a bacterial expression vector was used to express RBP4 in E.Coli as previously described (Shi et al., 2017). Apo-RBP4 (100 μg) was loaded with retinol in 0.2 mL of PBS by the addition of 100 μm radiolabeled retinol (American Radiolabeled Chemicals; vitamin A alcohol [3H(N)] Retinol-labeled, adjusted to 1 μCi/nmol specific activity by the addition of cold retinol) and incubating for 1 h at room temperature and then overnight at 4°C in light-protected tubes, as previously described (Shi et al., 2017). Stable NIH3T3 cells expressing either, RBPR2 or RBPR2 and LRAT were plated in 10 cm dishes. Cells were grown to 70% confluence, washed thrice with 1x PBS and incubated for 1 h in serum-free medium, at which point [³H]ROL-RBP4 was added for 60 min. Cells were washed thrice with 1x PBS and lysed in PBS containing 1% Nonidet P-40. Lysates were homogenized and transferred to scintillation tubes for scintillation counting. Parental NIH3T3 incubated with [³H]ROL-RBP4 served as controls. The RBP4-ROL binding and uptake assay was repeated thrice, using stable cells from a different passage.

Human RBP4 expression and purification from Escherichia coli was accomplished essentially as described previously (Shi et al., 2017). Briefly, human RBP4 (hRBP4) cDNA was cloned into a pET3a expression vector and expressed in BL-21 DE3 cells according to a standard protocol. Bacterial cells were harvested and lysed by osmotic shock. Insoluble material was pelleted by centrifugation, washed, and solubilized in 7M guanidine hydrochloride and 10 mM dithiothreitol. After overnight incubation, insoluble material was removed by ultracentrifugation, and the supernatant was used for the hRBP4 refolding procedure. hRBP4 was refolded by the dropwise addition of solubilized material into a mixture containing 150 μCi of [11,12-³H]ROL ([³H]ROL) (PerkinElmer Life Sciences) and non-radiolabeled ROL (Sigma) at a final concentration of 1 mm. Refolded holo-hRBP4 was dialyzed against 10 mM Tris/HCl buffer, pH 8.0, and loaded onto a DE53 anion exchange chromatography column (Whatman, Piscataway, NJ). Holo-hRBP4 was eluted with linear gradient of NaCl (0–1M) in 10 mM Tris/HCl buffer, pH 8.0. Collected fractions were examined by SDS-PAGE and UV-visible spectroscopy to ensure a proper protein/retinoid ratio. Fractions containing at least 90% holo-hRBP4 were pooled together and concentrated in a Centricon centrifugal filter device (cut-off 10,000 Da) (Millipore, Billerica, MA) to 5 mg/mL. [³H]ROL was quantified in a scintillation counter (Beckman Coulter, Indianapolis, IN). Holo-hRBP4 aliquots were stored at −80°C until used.

Total proteins from cells were extracted using the M-PER protein lysis buffer (ThermoScientific, Beverly, MA, United States) containing protease inhibitors (Roche, Indianapolis, IN, United States). Approximately 25 μg of total protein was electrophoresed on 4%–12% SDS-PAGE gels and transferred to PVDF membranes. Membranes were probed with primary antibodies against EGFR (1:1,000; ThermoFisher/Invitrogen, Waltham, MA), HSP90 (1:2500; Invitrogen, Waltham, MA), V5 (1:2500; Sigma/Millipore, Burlington, MA), Rbp4 (1:1,000; Proteintech/Fisher Scientific, Pittsburg, PA), or β-Actin (1:10,000, Sigma) in antibody buffer (0.2% Triton X-100, 2% BSA, 1X PBS). HRP-conjugated secondary antibodies (BioRad, Hercules, CA, United States) were used at 1:10,000 dilution. Protein expression was detected using a LI-COR Odyssey or ChemiDoc Bio-Rad system, and relative intensities of each band were quantified (densitometry) using ImageJ software version 1.49 and normalized to their respective loading controls. Each western blot analysis was repeated thrice.

Co-immunoprecipitation (Co-IP) assays were performed with exogenous applied human RBP4 protein in NIH3T3/LRAT cells stably expressing V5-tagged WT-RBPR2 or individual V5-tagged RBPR2-RBP4 mutants. Using a well-established Co-IP protocol that determined extracellular STRA6-RBP4 interactions, we added reduced serum medium (8 mL of OptiMEM) containing 12 μM of purified and crosslinked T7 tagged-RBP4 to the cells and incubate this reaction for 60 min (Kawaguchi et al., 2007). After binding purified T7 tagged-RBP4 protein conjugated with the cross-linker to cells expressing WT or mutant RBPR2, followed by ultraviolet (UV) cross-linking, and membrane solubilization, cells were collected, washed thrice with 1x PBS to remove any un-bound hRBP4. Total protein was isolated and subjected to co-immunoprecipitation analysis using an RBP4 antibody, followed by reciprocal western blotting for RBPR2 (using a V5 antibody).

Recombinant mouse RBP4 with 6XHis Tag was expressed E.coli expression system and extracted in Tris buffer with composition of 50 mM Tris-HCl, 1 M L-Arginine, 10% Glycerol, pH 8.0. The lysate was purified by nickel NTA column. The msRBP4 protein quality was monitored by western blot using anti His-tag antibody. The structural quality of the recombinant RBP4 protein was confirmed with Circular dichroism (CD) spectroscopy (Jasco 815 circular dichroism, Spectramax Gemini) (Micsonai et al., 2015). The mean residue ellipticity (θ), was calculated using the following formula. θ = S × m R w / 10 c l where S represents the CD signal in mθ, mRw represents the mean residue mass, c represents the concentration of the protein in mg/mL, and l represents the path length in cm. The percent change in molecules structure were calculated using BeStSel Secondary Structure Analysis to Protein Fold Prediction by CD Spectroscopy (https://bestsel.elte.hu), (see Supplementary Information Supplementary Materials S1–S5). The initial interaction quality of the recombinant RBP4 with msSTRA6, msRBPR2, and control peptides were checked with intrinsic tryptophan fluorescence assay. The peptides were diluted in various micromolar concentrations and the incubated with 3 μg RBP4 in room temperature for 5 min and excited at 290 nm and the emission was scanned from 300 nm to 400 nm wavelength. The data were normalized with the blank and peptide only conditions and plotted in GraphPad prism version 9.3. San Diego, CA, United States. (Supplementary Figure S5).

Purified RBP4 protein with >90% purity and 0.56 mg/mL concentration was immobilized on Biacore Sensor Chip CM5 (ITDD Biacore S200 Surface Plasmon Resonance instrument at University of Minnesota). The two-flow cell surface activated for using one as blank and other as test. Using Amine Coupling Kit (Cat. No. BR100050; Cytiva, Marlborough, MA, US) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), after surface activation, the purified RBP4 with immobilization buffer 10 mM Sodium acetate, pH 5.0, was immobilized with target of 1,200 Response Unit (RU) for achieving Rmax of 30RU in kinetic study. The reaction stopped and washed with Ethanolamine. The system was re-primed with running buffer PBST (phosphate-buffered saline solution with a 0.05% Tween20 detergent solution). The kinetic assay performed on the two flow cells, the blank was used as reference cell and the active cell with RBP4 was used for the binding study. The mouse and zebrafish RBPR2, mouse RBPR2 mutants affecting the “SYL” binding domain, and mouse STRA6 peptides (all containing the predicted RBP4 “SYL” binding residues) were chemically synthesized by Biomatik Corporation, Kitchener, ON, Canada. The peptides were serial diluted in running buffer with range of 0.8–26.6 μM and following parameter was run with contact time: 120 s, flowrate 30 μL/min, Dissociation time 300 s, Regeneration with Glycine-HCl, pH 2.5, contact time 30 s flowrate 30 μL/min and temperature 25°C. The program was run and non-specific binding on the reference cell subtracted bulk refractive index from the active sensorgram and analyzed for the association, dissociation and stabilization of the reads. The plot fitted with 1:1 binding program in Biacore™ Insight Evaluation Software, and the Graph, binding affinity plot, was plotted in GraphPad prism version 9.3.

Comparison of mouse (Ms) RBPR2 protein sequences to human (Hs) STRA6 and Ms STRA6 revealed several short amino acid segments with >40% amino acid homology, suggesting analogous roles for these residues in the function or structural integrity of these two proteins (Figures 1A–C) (Kawaguchi et al., 2008a; Kawaguchi et al., 2008b; Kawaguchi and Sun, 2010; Sun and Kawaguchi, 2011; Alapatt et al., 2013). Interestingly, a three amino acid consensus was found in the proposed RBP4 binding domain of Hs. And Ms. STRA6, which was also found to be partially conserved in mouse and zebrafish RBPR2. The proposed RBP4 binding residues in mouse RBPR2 correspond to amino acids Serine294, Tyrosine295, and Leucine296 (SYL) (Figure 1A), which have previously been shown to be required for vitamin A transport to the eye, in zebrafish (Shi et al., 2017; Lobo et al., 2018; Solanki et al., 2020).

To determine the importance of proposed RBP4 binding residues on mouse RBPR2, we first generated homology-based models of mouse RBPR2 and human STRA6 using the online server SWISS-MODEL (http://swissmodel.expasy.org/), using the cryogenic electron microscopy (cryoEM) structure of zebrafish STRA6 (PDB ID: 5sy1, Chain B) (Chen et al., 2016; van Zundert et al., 2016; Waterhouse et al., 2018), and human RBP4 (PDB ID: 2wqa, Chain E) (Figures 1B, C; Figures 2A, B). The models generated were then utilized in docking studies to analyze the STRA6-RBP4 and RBPR2-RBP4 protein-ligand interactions (docking program HADDOCK2.2) (Chen et al., 2016; van Zundert et al., 2016; Waterhouse et al., 2018). While the in silico binding models are assumptions, this analysis showed that the proposed and conserved residues SYL on mouse RBPR2 are part of an extracellular loop that likely plays a critical role in their interaction with RBP4 (Figures 2C–F) and thus stabilizing the complex interface with hydrophobic and hydrophilic contact. The 2D-Diagram shows the complex stabilized by the Conventional Hydrogen bond from Ser294 RBPR2, Pi-Cation and Pi-Alkyl from Tyr295 RBPR2 interacting with the Arg167 on RBP4. Leu296 on RBPR2 was not directly involved in the interaction interface, but could play an essential role in stabilizing the interaction with RBP4.

To study the importance of RBP4 binding residues on RBPR2, we used site-directed mutagenesis to individually alter these putative binding residues on mouse RBPR2. Using the wild-type (WT) RBPR2-pCDNA3.1-V5 tagged vector as a template, the polar amino acids (Ser294 and Tyr295) were mutated to hydrophobic amino acids (Ser294Ala and Tyr295Pro), while the hydrophobic amino acid (Leu296) was mutated to a polar amino acid (Leu296Ser) (Kawaguchi et al., 2008a; Kawaguchi et al., 2008b). WT-RBPR2 and individual RBPR2-mutants were transiently transfected into NIH3T3 cells, and at 72 h post-transfection, WT and mutant RBPR2 expressing cells were subjected to both immunostaining and western blot analysis using the V5-antibody. Confocal microscopy analyses revealed that similar to WT-RBPR2 protein, all three single RBPR2-RBP4 binding residue mutants trafficked properly to the plasma membrane in transiently transfected NIH3T3 cells (green = V5-tagged RBPR2) (Figure 3A). Western blot and densitometry analysis further revealed that like WT-RBPR2, all three single RBPR2-mutants were equally expressed (Figure 3B). To confirm the specific subcellular localization of WT and mutant RBPR2 proteins, we subjected the transfected cells to subcellular fractionation. This analysis confirmed that individual RBPR2-mutants, like WT-RBPR2 protein, localized predominantly within the plasma membrane fractions, with only two RBPR2-mutants showing minimal cytoplasmic retention (<2% of total fractionated protein), indicating that individual RBPR2 mutants, like WT-RBPR2, trafficked properly to the plasma membrane and was expressed in this fraction (Figures 3A, C).

To determine the importance of proposed RBP4 binding residues on RBPR2 for RBP4 binding and ROL transport, we generated stable NIH3T3/LRAT cells expressing WT-RBPR2 or individual RBPR2-mutants (Shi et al., 2017; Lobo et al., 2018). Using both V5 antibody, we first confirmed equal expression of all recombinant proteins in stable cells (Figure 4A). To determine the interaction of RBPR2 with exogenous RBP4, we performed Co-IP experiments. Stable NIH3T3/LRAT cells expressing WT or individual RBPR2 mutants were seeded in 10 cm dishes. Upon reaching ∼70% confluence, a reduced serum medium (8 mL of OptiMEM) containing 12 μM of purified RBP4 was added to the cells and incubated for 120 min. Cells were collected, and total protein was isolated and subjected to co-immunoprecipitation analysis using an RBP4 antibody, followed by reciprocal western blotting for RBPR2 (using a V5 antibody). While cells expressing WT-RBPR2 showed strong binding to exogenous RBP4 protein, individual mutant RBPR2-expressing cells showed decreased binding to RBPR2 (p < 0.005) (Figures 4A, B). To confirm this observation, individual stable cell lines were incubated with [³H]ROL-RBP4 and analyzed for their ability to uptake extracellular [³H]ROL-RBP4 at the 60-minute time point through Liquid Scintillation Counting (Shi et al., 2017; Lobo et al., 2018). This analysis showed that control cells (NIH3T3 and NIH3T3/LRAT cells) displayed insignificant levels of [³H]ROL-RBP4 uptake (Figure 4C). However, [³H]ROL was evident in cells expressing WT-RBPR2 with co-expressed LRAT (Figure 4C). In contrast, individual RBPR2-mutant expressing cells showed significantly reduced ability (<81% decreased activity compared to WT-RBPR2; p < 0.005) to uptake [³H]ROL-RBP4, indicating that the amino acids Ser294, Tyr295, and Leu296 likely encompass the RBP4 binding residues on RBPR2 that would be crucial for ROL transport (Figure 1). Based on proper membrane trafficking of mutant RBPR2 protein (Figure 3A), but with decreased RBP4 binding (Figures 4A, B) and [³H]ROL-RBP4 uptake capabilities (Figure 4C), indicates the importance of these residues on RBPR2 for extracellular RBP4 interaction/binding, which is in turn critical for ROL transport.

Surface Plasmon Resonance (SPR) is a common technique used to study protein-ligand interactions. SPR can measure the binding affinities and association/dissociation kinetics of protein to ligand complexes in real-time. The interaction levels measured in Response Units (RU), and real-time plot sensorgram displays the dynamics of the analysis. The purified mouse Retinol Binding Protein, RBP4 (Supplementary Figure S1), was immobilized as the ligand, and various concentrations of the SYL motif containing peptides as analytes were run to measure SPR affinity and kinetics. The analytes examined were mouse RBPR2, zebrafish RBPR2, and mouse STRA6, known to interact with the mouse RBP4 ligand (Supplementary Figures S2–S5). To determine the K _d (ligand concentration in which half of the total receptor sites are occupied), the site-specific binding fitting model described below was used. Y = B m a x ∗ X K d + X where B_max represents the maximum number of binding sites (Response Unit/RU), X represents the analyte concentration, and Y represents the binding affinity (Response Unit). Interestingly, the binding affinity of mouse RBPR2 to RBP4 approximates to the affinity of mouse STRA6 peptides to RBP4. The K _d of WT-msRBPR2 peptide with msRBP4 was 25.42 ± 6.01 μM, and B_max was 183.17 ± 25.66 μM (mean ± S.E). The K _d of msSTRA6 peptide with msRBP4 was 26.73 ± 5.67 μM, and B_max was 178.45 ± 22.26 (mean ± S.E) (Figures 5, 6). The difference between the binding affinities and dissociation rate (K _off) of msRBPR2 and msSTRA6 to msRBP4 was not statistically significant (p < 0.99) in an unpaired t-test, suggesting similar K _d values and binding affinities of these two proteins for its extracellular ligand (Supplementary Figure S6). SPR analysis was then performed on SYL mutant mouse RBPR2 with its physiological ligand RBP4. This analysis showed that RBPR2 mutants (S294A and Y295P), had higher K _d values of 89.33 μM and 34.91 μM respectively, while the RBPR2 mutant (L296S) had a lower K _d value of 21.30 μM, compared to K _d value of 25.42 μM for WT-RBPR2, dissociation rate (K _off) for mutant msRBPR2 was significantly lower, suggesting a tighter bond formation between the RBP4 protein and mutant peptides (Figures 7A–C; Supplementary Figure S7).