kinetic assay setup for analyzing the kinetic parameters of rbpr2 and stra6 interaction with vitamin a receptor

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.

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.

kinetic-assay-setup-for-analyzing-kinetic-parameters-rbpr2-stra6

Research

JoVE Journal

Biochemistry

38 visualizações. Para configurar o ensaio cinético para os peptídeos RBPR2 e STRA6, dilua em série os peptídeos RBPR2, RBPR2 e STRA6 mutantes do camundongo no tampão de corrida. Abra o software de controle e selecione o assistente Kinetic SOAR Affinity. Na caixa de diálogo da sequência de injeção, selecione Caminho de fluxo 2.1 com a célula de fluxo 2 como ativa e a célula de fluxo 1 como referência.Na caixa de diálogo de configuração, marque a opção e...