spectrophotometry methodology to quantify the gpcr-11-cis retinal protein complex in retinal lysates

Investigating Vitamin A Receptor Kinetics and Function for Retinoid Homeostasis

Dietary obtained Vitamin A/all-trans retinol/ROL is an important component playing a role in visual function1,2. The chromophore 11-cis retinal, a metabolite of dietary vitamin A, binds to the G protein-coupled receptor (GPCR) opsin to generate the retinylidene protein, rhodopsin, in the photoreceptors. When light falls on the eye, the configuration of rhodopsin undergoes a fundamental change via the conversion of its 11-cis-retinal component to the all-trans-retinal. This configuration change triggers a phototransduction cascade within the rod photoreceptors, converting light into an electrical signal, which is transmitted to the visual cortex in the brain via the optic nerve3,4,5,6,7,8,9,10. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn causes opsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor OS degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis, which can cause blindness3,10.
All-trans retinol is the fundamental transport form of dietary vitamin A and it is the source from which all functional retinoids and dietary vitamin A metabolites are derived. The liver serves as the primary organ for dietary vitamin A storage. Hepatic retinol is transported via the serum as its complex with retinol-binding protein 4 (RBP4). RBP4, primarily expressed in the liver, forms a holo-complex with the retinol substrate and transthyretin (TTR), which enters the circulation11,12,13,14,15,16,17. The report of a cell surface receptor for RBP4 in the 1970s led to the hypothesis of membrane transport proteins aiding the transport of retinoids in and out of cells. The cell surface receptor for RBP4-bound retinol (RBP4-ROL) was identified as STimulated by Retinoic Acid 6 Retinol (STRA6) in the retinal pigment epithelium (RPE) of the eye. STRA6 binds to the circulatory holo-RBP4 complex and shuttles the RBP4-bound retinol across the RPE to be utilized by photoreceptors18,19. Mutations in STRA6 can lead to a myriad of diseases and phenotypes associated with reduced ocular ROL concentrations. STRA6 mutations during development can lead to anophthalmia, microphthalmia, and other non-ocular symptoms that overlap with phenotypes associated with Matthew-Wood syndrome20,21,22,23,24,25,26,27. STRA6 is expressed in different organs and tissues, such as the RPE in the eye, but not in all tissues26,27. Although the primary site of retinoid storage is the liver, STRA6 is not expressed in the liver.
Alapatt and colleagues discovered that the Retinol Binding Protein 4 Receptor 2 (RBPR2) bound RBP4 with high affinity and was responsible for the uptake of RBP4-bound retinol in the liver, similar to STRA6 in the RPE28. RBPR2 has been reported to share structural homology with STRA629,30,31. RBP4 is proposed to bind to residues S294, Y295, and L296 on RBPR2, an amino acid-binding domain partially conserved between RBPR2 and STRA6 as well29,30,31. From these studies, vitamin A membrane receptors such as STRA6 and RBPR2, which contain one or more extracellular binding residues/domains, are proposed to interact with circulatory RBP4-ROL. Membrane receptors, therefore, play an important role in receptor binding to circulatory RBP4 for ROL internalization into target tissues, such as the liver and eye.
In the first part of this study, we utilized Surface Plasmon Resonance (SPR) to investigate the interaction of two vitamin A membrane receptors (RBPR2 and STRA6) with their physiological ligand RBP431. The binding affinities and association/dissociation kinetics of protein to ligand complexes can be measured in real time by using SPR. This methodology aimed to provide critical kinetic, structural, and biochemical information on RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency31,32. As mentioned above, circulatory ROL is internalized into the RPE via STRA6 to generate the chromophore 11-cis retinal, which binds to opsin to generate the retinylidene protein, rhodopsin, in photoreceptors33. We used spectrophotometry methodologies to quantify the GPCR-opsin and its ligand 11-cis retinal complex in murine retinal lysates, which is critical for understanding mechanisms of reduced retinylidene protein, rhodopsin, in Retinitis Pigmentosa or Leber Congenital Amaurosis ocular pathological states34. In general, these protocols can be applied to study in vitro the physiological consequences of mutant RBP4, STRA6, or RBPR2 in influencing systemic and ocular vitamin A homeostasis or the impact of mutant rhodopsin or retinoid cycle proteins on visual function35,36.

Author Spotlight: Unraveling Vitamin A Transport Mechanisms &amp;#8212; Linking Liver Receptors to Vision Health Through RBPR2 and RBP4 Interactions

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

Our research investigates how vitamin A homeostasis is regulated, focusing on physiological interaction between retinal binding protein receptor two, RRBPR2, and its ligand, RRBP4, for whole-body vitamin A transport. We also explore how 11-cis retinal binds to rhodopsin, which is crucial for phototransduction in vision. We use two known techniques to establish a link between vitamin A storage and distribution by liver receptors, and the impact of interrupting this mechanism in the wellness of vision by accumulating disease-causing appoxins.Our protocol utilizes two techniques, focusing on distant communication between the liver and the eye for vitamin A transport. Our protocol makes it more evident and easier to correlate the disease conditions caused by the dietary or hereditary issues. The blood serum protein and factors stabilize the communication between the retinol binding protein receptor and its ligand, RRBP4.Finding all the relevant mechanisms is complicated, yet essential for the next part in answering some unexplored areas. In the future, our focus will be on translating the research protocol, studying the correlation of disease phenotypes in human patient samples, and looking for genetic variants of vitamin A receptors in disease.

Spectrophotometry Methodology to Quantify the GPCR-11-cis Retinal Protein Complex in Retinal Lysates

To begin, enucleate the eye globes of the euthanized mouse and homogenize the eyes in an ice cold buffer. Centrifuge the homogenate at 16, 000 G for 15 minutes at four degrees Celsius. Discard the supernatant containing water soluble proteins and other contaminants.Then resuspend and solubilize the pelleted membrane and membrane proteins in a buffer for one hour at four degrees Celsius on a rotating platform. Afterward, centrifuge the resolubilized membrane fraction for one hour at 16, 000 G at four degrees Celsius. Mix the supernatant with 30 to 50 microliters of Rho1D4 antibody beads, and blank immunoglobulin G antibody beads in two separate independent samples and incubate for one hour at four degrees Celsius on a rotating platform.Using a magnetic stand, separate the pull-down by collecting the beads. Discard the supernatant and wash the beads with a high and low salt buffer containing Bis-tris propane, sodium chloride, and N-dodecyl-beta-D-maltoside. To elute the rhodopsin protein, incubate the beads for five to 10 minutes at room temperature with a 65 microliter buffer of the given composition.Then using a rectangular sub-micro 50 microliter quartz cubit with a two millimeter aperture and 10 millimeter path length, analyze the eluate for absorbance from 200 nanometers to 800 nanometers in a fast scan setting with a spectrophotometer. To validate and determine the quality, expose the eluate to bright light for one minute. Then repeat the absorbance measurement.Subtract the blank absorbance and calculate the ratio of the analyzed absorbent spectra. Then plot the blank subtracted absorbent spectra, and calculate the free opsin value. Afterward, using Beer-Lambert law, calculate the concentration of rhodopsin, then calculate the concentration of ligand-free opsin, and estimate the difference in concentration for apo-opsin, and ligand-free opsin in amount and percentage.

spectrophotometry-methodology-to-quantify-gpcr-11-cis-retinal-protein

Research

JoVE Journal

Biochemistry

63 Views. To begin, enucleate the eye globes of the euthanized mouse and homogenize the eyes in an ice cold buffer. Centrifuge the homogenate at 16, 000 G for 15 minutes at four degrees Celsius. Discard the supernatant containing water soluble proteins and other contaminants.Then resuspend and solubilize the pelleted membrane and membrane proteins in a buffer for one hour at four degrees Celsius on a rotating platform. Afterward, centrifuge the resolubilized membrane fraction for one hour at 16,...

Video: Spectrophotometry Methodology to Quantify the GPCR-11-cis Retinal Protein Complex in Retinal Lysates

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and generating the retinylidene protein for visual function. RBP4-ROL is the complex of ROL with retinol-binding protein 4 (RBP4), which is present in the blood. Two membrane receptors, Retinol Binding Protein 4 Receptor 2 (RBPR2) in the liver and STimulated by Retinoic Acid 6 Retinol (STRA6) in the eye, bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Establishing methods to investigate receptor-ligand kinetics is essential in understanding the physiological function of vitamin A receptors for retinoid homeostasis. Using Surface Plasmon Resonance (SPR) assays, we can analyze the binding affinities and kinetic parameters of vitamin A membrane receptors with its physiological ligand RBP4. 
These methodologies can reveal new structural and biochemical information of RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency. In the eye, internalized ROL is metabolized to 11-cis retinal, the visual chromophore that binds to opsin in photoreceptors to form the retinylidene protein, rhodopsin. The absorbance of light causes the cis-to-trans isomerization of 11-cis retinal, inducing conformational changes in rhodopsin and the subsequent activation of the phototransduction cascade. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn can cause rhodopsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor outer segment degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis. 
Therefore, spectrophotometric methodologies to quantify the G protein-coupled receptor opsin-11-cis retinal complex in the retina are critical for understanding mechanisms of retinal cell degeneration in the above-mentioned pathological states. With these comprehensive methodologies, investigators will be able to better assess dietary vitamin A supply in maintaining systemic and ocular retinoid homeostasis, which is critical for generating and maintaining retinylidene protein concentrations in photoreceptors, which is critical for sustaining visual function in humans.

Here, we describe two quantitative methods for studying the protein-ligand interactions of vitamin A membrane receptors and photoreceptor opsin with their respective physiological ligands.

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and ...

SPR Analysis to Determine the Binding Affinities of Vitamin A Membrane Receptors with Their Physiological Ligand RBP4

Begin by immobilizing the purified msRBP4 protein on the sensor chip. To do so, go to the Run menu, choose the wizard, and proceed with the immobilization option. Next, dilute the ligand in immobilization buffer to a concentration of 10 micrograms per milliliter, and aliquot the coupling reagents into vials.Now, open the Immobilization Setup wizard window. Select the CM5 chip and immobilize flow cell 2 options. Then set the target level to 1200 response units for an R maximum of 30 response units in the kinetic study.Choose ethanolamine as the wash solution. Enter the name RBP4, and click Next. In the Rack Position dialogue box, click Eject Rack.Remove the rack. Place the vials containing the specified volumes of each reagent. And then insert the rack with the sample vials.After that, click OK and Next. Finally, in the Prepare Run Protocol dialogue, click Start and then Save.

Kinetic Assay Setup for Analyzing the Kinetic Parameters of RBPR2 and STRA6 Interaction with Vitamin A Receptor

To set up the Kinetic assay for RBPR2 and STRA6 peptides serially dilute the mouse RBPR2, mutant RBPR2 and STRA6 peptides in running buffer. Open the control software and select the Kinetic SOAR Affinity wizard. In the injection sequence dialogue, select Flow path 2.1 with Flow cell 2 as active and Flow cell 1 as reference.In the setup dialogue, check the run startup cycles option, input the solution as buffer, specify the number of cycles and click next. Now go to the injection parameters dialogue and enter sample parameters such as contact time of 120 seconds, flow rate of 30 microliters per minute, and dissociation time of 300 seconds. For regeneration parameters, set the regeneration solution to glycine hydrochloric acid with a contact time of 30 seconds and a flow rate of 30 microliters per minute, then click next.In the samples dialogue enter the peptides names, molecular weights, and concentrations, then click next. In the rack position dialogue assign the diluted peptides as individual 120 microliter samples with various concentrations from one to 100 micromolar to the designated positions on the rack. Click eject rack and insert all the vials.Afterward, click next and then in the prepare run protocol dialogue click start. Enter a file name and click save. Now in the software, click the start menu and select a valuation.Open the file then from the dropdown selection menu choose FC-2.1 to analyze the reference cell blank subtracted active sensorgram for the association and dissociation phases of msRBP4, mutant msRBPR2, and msSTRA6 peptides. Next, click the dropdown Kinetics or Affinity button and choose surface bound. In the select curves dialogue, check include column in the table, and click next.Now go to the select data dialogue, view the blank subtracted sensorgrams. Click Kinetics and Affinity. Keep the default parameters and click fit for curve fitting.Inspect the report tab for equilibrium dissociation constant Kd, association rate Ka or Kon and dissociation rate Kd or Koff. Save the Kinetics data sensorgram in text tab to limited format and use the values to plot graphs.