title: Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways.
abstract: Resolution limitations of optical systems are major obstacles for determining whether proteins are enriched within cell compartments. Here we use an approach to determine the degree of membrane protein ciliary enrichment that quantitatively accounts for the differences in sampling of the ciliary and apical membranes inherent to confocal microscopes. Theory shows that cilia will appear more than threefold brighter than the surrounding apical membrane when the densities of fluorescently labeled proteins are the same, thus providing a benchmark for ciliary enrichment. Using this benchmark, we examined the ciliary enrichment signals of two G protein-coupled receptors (GPCRs)-the somatostatin receptor 3 and rhodopsin. Remarkably, we found that the C-terminal VxPx motif, required for efficient enrichment of rhodopsin within rod photoreceptor sensory cilia, inhibited enrichment of the somatostatin receptor in primary cilia. Similarly, VxPx inhibited primary cilium enrichment of a chimera of rhodopsin and somatostatin receptor 3, where the dual Ax(S/A)xQ ciliary targeting motifs within the third intracellular loop of the somatostatin receptor replaced the third intracellular loop of rhodopsin. Rhodopsin was depleted from primary cilia but gained access, without being enriched, with the dual Ax(S/A)xQ motifs. Ciliary enrichment of these GPCRs thus operates via distinct mechanisms in different cells.
authors.affiliation: Center for Vision Research, Department of Ophthalmology, State University of New York Upstate Medical University, Syracuse, NY 13210.|Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, NY 13210.
authors.forename: Ivayla I
authors.initials: II
authors.lastname: Geneva
authors.affiliation: Center for Vision Research, Department of Ophthalmology, State University of New York Upstate Medical University, Syracuse, NY 13210.
authors.forename: Han Yen
authors.initials: HY
authors.lastname: Tan
authors.affiliation: Center for Vision Research, Department of Ophthalmology, State University of New York Upstate Medical University, Syracuse, NY 13210 calvertp@upstate.edu.|Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, NY 13210.|Department of Neuroscience and Physiology, State University of New York Upstate Medical University, Syracuse, NY 13210.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Calvert
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002923:Cilia / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D017481:Receptors, Somatostatin / Q000378:metabolism*; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism* / Q000502:physiology; D012243:Rhodopsin / Q000378:metabolism*
chemical_list: D043562:Receptors, G-Protein-Coupled; D017481:Receptors, Somatostatin; C103631:somatostatin receptor 3; D012243:Rhodopsin