Name: Author Spotlight: Unraveling Vitamin A Transport Mechanisms &#8212; Linking Liver Receptors to Vision Health Through RBPR2 and RBP4 Interactions
Uploaded: 2024-10-04T14:10:00
Description: Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and ...

The authors thank Dr. Beata Jastrzebska, Ph.D. (Department of Pharmacology, Case Western Reserve University, OH) for her advice on the rhodopsin absorbance protocol. This work was supported by an NIH-NEI grant (EY030889 and 3R01EY030889-03S1) and, in part, by the University of Minnesota start-up funds to G.P.L.

1. Surface plasmon resonance (SPR) methodology
<ol>
	<li>Preparation and purification of mouse RBP4 protein
	<ol>
		<li>Design primers to generate full-length mouse RBP4 (msRBP4) cDNA (NCBI Reference Sequence: NM_001159487.1). Clone the msRBP4 cDNA into a pET28a His-tag Kanamycin-resistant expression vector and express in BL-21 DE3 cells (Table of Materials).</li>
		<li>Transform the BL-21 DE3 competent cells (according to the manufacturer&#39;s protocol) with ligated...

Investigating Vitamin A Receptor Kinetics and Function for Retinoid Homeostasis

<ol>
	<li>Dowling, J. E., Wald, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+A+deficiency+and+night+blindness.">Vitamin A deficiency and night blindness.</a> Proc Natl Acad Sci U S A. 44 (7), 648-6618 (1958).</li><li>Dowling, J. E., Wald, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biological+function+of+vitamin+A+acid.">The biological function of vitamin A acid.</a> Proc Natl Acad Sci U S A. 46 (5), 587-608 (1960).</li><li>Kiser, P. D., Palczewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+and+retinal+diseases.">Retinoids and retinal diseases.</a> Annu Rev Vis Sci. 2 (1), 197-234 (2016).</li><li>Harrison, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+involved+in+the+intestinal+absorption+of+dietary+vitamin+A+and+provitamin+A+carotenoids.">Mechanisms involved in the intestinal absorption of dietary vitamin A and provitamin A carotenoids.</a> Biochim Biophys Acta. 1821 (1), 70-77 (2012).</li><li>Harrison, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotenoids,+&#946;-apocarotenoids,+and+retinoids:+the+long+and+the+short+of+it.">Carotenoids, &#946;-apocarotenoids, and retinoids: the long and the short of it.</a> Nutrients. 14 (7), 1411 (2022).</li><li>Li, Y., Wongsiriroj, N., Blaner, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifaceted+nature+of+retinoid+transport+and+metabolism.">The multifaceted nature of retinoid transport and metabolism.</a> Hepatobiliary Surg Nutr. 3 (3), 126-139 (2014).</li><li>O'Byrne, S. M., Blaner, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinol+and+retinyl+esters:+biochemistry+and+physiology.">Retinol and retinyl esters: biochemistry and physiology.</a> J Lipid Res. 54 (7), 1731-1743 (2013).</li><li>Blomhoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+and+metabolism+of+vitamin+A.">Transport and metabolism of vitamin A.</a> Nutr Rev. 52 (2 Pt 2), S13-S23 (2009).</li><li>Chelstowska, S., Widjaja-Adhi, M. A. K., Silvaroli, J. A., Golczak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+for+vitamin+A+uptake+and+Storage+in+Vertebrates.">Molecular basis for vitamin A uptake and Storage in Vertebrates.</a> Nutrients. 8 (11), 676 (2016).</li><li>Martin Ask, N., Leung, M., Radhakrishnan, R., Lobo, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+A+transporters+in+visual+function:+A+mini+review+on+membrane+receptors+for+dietary+vitamin+A+uptake,+storage,+and+transport+to+the+eye.">Vitamin A transporters in visual function: A mini review on membrane receptors for dietary vitamin A uptake, storage, and transport to the eye.</a> Nutrients. 13 (11), 3987 (2021).</li><li>Yamamoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+transthyretin+(TTR)+and+retinol-binding+protein+(RBP)+in+the+uptake+of+retinol+by+primary+rat+hepatocytes.">Interactions of transthyretin (TTR) and retinol-binding protein (RBP) in the uptake of retinol by primary rat hepatocytes.</a> Exp Cell Res. 234 (2), 373-378 (1997).</li><li>Chen, C. C., Heller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+of+retinol+and+retinoic+acid+from+serum+retinol-binding+protein+by+retinal+pigment+epithelial+cells.">Uptake of retinol and retinoic acid from serum retinol-binding protein by retinal pigment epithelial cells.</a> J Biol Chem. 252 (15), 5216-5221 (1977).</li><li>Gj&#248;en, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+takes+up+retinol-binding+protein+from+plasma.">Liver takes up retinol-binding protein from plasma.</a> J Biol Chem. 262 (23), 10926-10930 (1987).</li><li>Blomhoff, R., Norum, K. R., Berg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+uptake+of+[3H]retinol+bound+to+the+serum+retinol+binding+protein+involves+both+parenchymal+and+perisinusoidal+stellate+cells.">Hepatic uptake of [3H]retinol bound to the serum retinol binding protein involves both parenchymal and perisinusoidal stellate cells.</a> J Biol Chem. 260 (25), 13571-13575 (1985).</li><li>Kanai, M., Raz, A., Goodman, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinol-binding+protein:+The+transport+protein+for+vitamin+A+in+human+plasma.">Retinol-binding protein: The transport protein for vitamin A in human plasma.</a> J Clin Invest. 47 (9), 2025-2044 (1968).</li><li>Quadro, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+extrahepatic+retinol+binding+protein+in+the+mobilization+of+retinoid+stores.">The role of extrahepatic retinol binding protein in the mobilization of retinoid stores.</a> J Lipid Res. 45 (11), 1975-1982 (2004).</li><li>Blaner, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STRA6,+a+cell-surface+receptor+for+retinol-binding+protein:+The+plot+thickens.">STRA6, a cell-surface receptor for retinol-binding protein: The plot thickens.</a> Cell Metab. 5 (3), 164-166 (2007).</li><li>Kawaguchi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+receptor+for+retinol+binding+protein+mediates+cellular+uptake+of+vitamin+A.">Membrane receptor for retinol binding protein mediates cellular uptake of vitamin A.</a> Science. 315 (5813), 820-825 (2007).</li><li>Kawaguchi, R., Zhong, M., Kassai, M., Ter-Stepanian, M., Sun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STRA6-catalyzed+vitamin+A+influx,+Efflux,+and+Exchange.">STRA6-catalyzed vitamin A influx, Efflux, and Exchange.</a> J. Membr. Biol. 245 (11), 731-745 (2012).</li><li>Pasutto, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+STRA6+cause+a+broad+spectrum+of+malformations+including+anophthalmia,+congenital+heart+defects,+diaphragmatic+hernia,+alveolar+capillary+dysplasia,+lung+hypoplasia,+and+mental+retardation.">Mutations in STRA6 cause a broad spectrum of malformations including anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and mental retardation.</a> Am J Hum Genet. 80 (3), 550-560 (2007).</li><li>Golzi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matthew-Wood+Syndrome+is+caused+by+truncating+mutations+in+the+retinol-binding+protein+receptor+gene+STRA6.">Matthew-Wood Syndrome is caused by truncating mutations in the retinol-binding protein receptor gene STRA6.</a> Am J Hum Genet. 80 (14), 1179-1187 (2007).</li><li>Isken, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RBP4+disrupts+vitamin+A+uptake+homeostasis+in+a+STRA6-deficient+animal+model+for+Matthew-Wood+Syndrome.">RBP4 disrupts vitamin A uptake homeostasis in a STRA6-deficient animal model for Matthew-Wood Syndrome.</a> Cell Metab. 7 (3), 258-268 (2008).</li><li>Ruiz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoid+content,+visual+responses,+and+ocular+morphology+are+compromised+in+the+retinas+of+mice+lacking+the+retinol-binding+protein+receptor,+STRA6.">Retinoid content, visual responses, and ocular morphology are compromised in the retinas of mice lacking the retinol-binding protein receptor, STRA6.</a> Investig Opthalmol Vis Sci. 53 (6), 3027-3039 (2012).</li><li>Kelly, M., von Lintig, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STRA6:+Role+in+cellular+retinol+uptake+and+efflux.">STRA6: Role in cellular retinol uptake and efflux.</a> Hepatobiliary Surg Nutr. 4 (4), 229 (2015).</li><li>Berry, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+STRA6+receptor+is+essential+for+retinol-binding+protein-induced+insulin+resistance+but+not+for+maintaining+vitamin+A+homeostasis+in+tissues+other+than+the+eye.">The STRA6 receptor is essential for retinol-binding protein-induced insulin resistance but not for maintaining vitamin A homeostasis in tissues other than the eye.</a> J Biol Chem. 288 (34), 24528-24539 (2013).</li><li>Amengual, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STRA6+is+critical+for+cellular+vitamin+A+uptake+and+homeostasis.">STRA6 is critical for cellular vitamin A uptake and homeostasis.</a> Hum Mol Genet. 23 (20), 5402-5417 (2014).</li><li>Steinhoff, J. S., Lass, A., Schupp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoid+homeostasis+and+beyond:+How+retinol+binding+protein+4+contributes+to+health+and+disease.">Retinoid homeostasis and beyond: How retinol binding protein 4 contributes to health and disease.</a> Nutrients. 14 (6), 1236 (2022).</li><li>Alapatt, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+retinol+transporter+and+receptor+for+serum+retinol-binding+protein+(RBP4).">Liver retinol transporter and receptor for serum retinol-binding protein (RBP4).</a> J Biol Chem. 288 (2), 1250-1265 (2013).</li><li>Radhakrishnan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+lacking+the+systemic+vitamin+A+receptor+RBPR2+show+decreased+ocular+retinoids+and+loss+of+visual+function.">Mice lacking the systemic vitamin A receptor RBPR2 show decreased ocular retinoids and loss of visual function.</a> Nutrients. 14 (12), 2371 (2022).</li><li>Solanki, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+binding+domain+in+the+Rbpr2+receptor+is+required+for+vitamin+A+transport,+ocular+retinoid+homeostasis,+and+photoreceptor+cell+survival+in+zebrafish.">A functional binding domain in the Rbpr2 receptor is required for vitamin A transport, ocular retinoid homeostasis, and photoreceptor cell survival in zebrafish.</a> Cells. 9 (5), 1099 (2020).</li><li>Radhakrishnan, R., Leung, M., Solanki, A. K., Lobo, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+extracellular+RBP4+ligand+binding+domain+on+the+RBPR2+receptor+for+vitamin+A+transport.">Mapping of the extracellular RBP4 ligand binding domain on the RBPR2 receptor for vitamin A transport.</a> Front Cell Dev Biol. 11 (11), 1105657 (2023).</li><li>Ramkumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vitamin+A+transporter+STRA6+adjusts+the+stoichiometry+of+chromophore+and+opsins+in+visual+pigment+synthesis+and+recycling.">The vitamin A transporter STRA6 adjusts the stoichiometry of chromophore and opsins in visual pigment synthesis and recycling.</a> Hum Mol Genet. 31 (4), 548-560 (2022).</li><li>Tian, H., Sakmar, T. P., Huber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+energetics+of+chromophore+binding+in+the+visual+photoreceptor+rhodopsin.">The energetics of chromophore binding in the visual photoreceptor rhodopsin.</a> Biophys J. 113 (1), 60-72 (2017).</li><li>Fan, J., Rohrer, B., Frederick, J. M., Baehr, W., Crouch, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rpe65-/-+and+Lrat-/-+mice:+comparable+models+of+leber+congenital+amaurosis.">Rpe65-/- and Lrat-/- mice: comparable models of leber congenital amaurosis.</a> Invest Ophthalmol Vis Sci. 49 (6), 2384-2389 (2008).</li><li>Breen, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+functional+human+vitamin+A+transporter/RBP+receptor+(STRA6)+for+structure+determination.">Production of functional human vitamin A transporter/RBP receptor (STRA6) for structure determination.</a> PLoS One. 10 (3), e0122293 (2015).</li><li>Kawaguchi, R., Zhong, M., Sun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analyses+of+retinol+transport+by+the+membrane+receptor+of+plasma+retinol+binding+protein.">Real-time analyses of retinol transport by the membrane receptor of plasma retinol binding protein.</a> J Vis Exp. 28 (71), e50169 (2013).</li><li>Shi, Y., Obert, E., Rahman, B., Rohrer, B., Lobo, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinol+binding+protein+receptor+2+(Rbpr2)+is+required+for+photoreceptor+outer+segment+morphogenesis+and+visual+function+in+zebrafish.">The retinol binding protein receptor 2 (Rbpr2) is required for photoreceptor outer segment morphogenesis and visual function in zebrafish.</a> Sci Rep. 7 (1), 16207 (2017).</li></ol>

Critical steps in the protocol 
SPR Methodology 
In silico modeling and docking analysis: The predicted structure of RBPR2 (https://alphafold.ebi.ac.uk/entry/Q9DBN1) and STRA6, and the known structure for&#160;msRBP4 PDB database (RSCB PDB ID: 2wqa), should be used for docking study29,31. Additionally, in vitro methods (cell culture) should be used to confirm the interaction of the putative bi...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-D Quant Kit</td>
			<td>Cytiva</td>
			<td>80648356</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine Coupling Kit</td>
			<td>Cytiva</td>
			<td>BR100050</td>
			<td></td>
		</tr>
		<tr>
			<td>Biacore evaluation software</td>
			<td>Biacore S200</td>
			<td>Version 1.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Biacore Sensor chip CM5</td>
			<td>Cytiva</td>
			<td>BR100530</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis tris propane</td>
			<td>Sigma</td>
			<td>B6755-25G</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td>BL21 DE3 competent cells</td>
			<td>Thermo Scientific</td>
			<td>EC0114</td>
			<td></td>
		</tr>
		<tr>
			<td>CD spectrophotometer</td>
			<td>Jasco</td>
			<td>J-815 Spectropolarimeter</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine HCL</td>
			<td>Fisher Bioreagents</td>
			<td>BP381-1</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism&#160;</td>
			<td></td>
			<td></td>
			<td>Model fitting, data analysis</td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Fisher Bioreagents</td>
			<td>BP1426-500</td>
			<td></td>
		</tr>
		<tr>
			<td>n-dodecyl-&#946;-d-maltoside (DDM)</td>
			<td>EMD Millipore</td>
			<td>324355-1GM</td>
			<td>2-20 mM</td>
		</tr>
		<tr>
			<td>pET28a His-tag Kanamycin-resistant expression vector&#160;</td>
			<td>Addgene</td>
			<td>69864-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid purification kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>Rho1D4 MagBeads</td>
			<td>CubeBiotech</td>
			<td>33299</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide-A_Lyzer 10K dialysis cassette</td>
			<td>Thermo Scientific</td>
			<td>66810</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Fisher Bioreagents</td>
			<td>BP337-500</td>
			<td>0.05%</td>
		</tr>
		<tr>
			<td>UV vis Spectrophotometer</td>
			<td>Agilent&#160;</td>
			<td>Cary 60 UV-Vis</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptide name</td>
			<td>Peptide sequence</td>
			<td>HPLC-purity</td>
			<td>Mass Spec</td>
		</tr>
		<tr>
			<td>Mouse Rbpr2 (42)</td>
			<td>HVRDKLDMFEDKLESYLTHM 
			NETGTLTPIILQVKELISVTKG</td>
			<td>92.14%</td>
			<td>Conforms</td>
		</tr>
		<tr>
			<td>Mouse Stra6 (40)</td>
			<td>SVVPTVQKVRAGINTDVSYL 
			LAGFGIVLSEDRQEVVELVK</td>
			<td>90.84%</td>
			<td>Conforms</td>
		</tr>
		<tr>
			<td>Mouse Rbpr2 mutant S294A (42)</td>
			<td>HVRDKLDMFEDKLEAYLTHM 
			NETGTLTPIILQVKELISVTKG</td>
			<td>0.92%</td>
			<td>Conforms</td>
		</tr>
	</tbody>
</table>

methodology for studying interactions of vitamin a membrane receptors and opsin protein with their ligands in generating the retinylidene protein

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.

Author Spotlight: Unraveling Vitamin A Transport Mechanisms &#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.

Quantitative methods are described to study protein-ligand interactions of vitamin A membrane receptors and photoreceptor opsin with their respective physiological ligands. The recombinant mouse RBP4 should be expressed in E. coli and the purified protein used as a conjugated ligand on a SPR Chip. The chemically synthesized RBPR2, STRA6, and mutant S294A RBPR2 &#34;SYL motif RBP4 interacting extracellular site&#34; of a ~40 amino acid peptide is used as analyte at various concentrations to measure the kinetics o...

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 ...

methodology-for-studying-interactions-vitamin-membrane-receptors