Abstract |
OA1 (GPR143) is a pigment cell-specific intracellular glycoprotein consisting of 404 amino acid residues that is mutated in patients with ocular albinism type 1, the most common form of ocular albinism. While its cellular localization is suggested to be endolysosomal and melanosomal, the physiological function of OA1 is currently unclear. Recent reports predicted that OA1 functions as a G protein coupled receptor (GPCR) based on its weak amino acid sequence similarity to known GPCRs, and on demonstration of GPCR activity in OA1 mislocalized to the plasma membrane. Because mislocalization of proteins is often caused by or induces defects in their proper folding/assembly, the significance of these studies remains unclear. A characteristic feature of GPCRs is a seven transmembrane domain structure. We analyzed the membrane topology of OA1 properly localized to intracellular lysosomal organelles in COS-1 cells. To accomplish this analysis, we established experimental conditions that allowed selective permeabilization of the plasma membrane while leaving endolysosomal membranes intact. Domains were mapped by the insertion of a hemagglutinin (HA) tag into the predicted cytosolic/ luminal regions of OA1 molecule and the accessibility of tag to HA antibody was determined by immunofluorescence. HA-tagged lysosome associated membrane protein 1 (LAMP1), a type I membrane protein, was employed as a reporter for selective permeabilization of the plasma membrane. Our results show experimentally that the C-terminus of OA1 is directed to the cytoplasm and that the protein spans the intracellular membrane 7 times. Thus, OA1, properly localized intracellularly, is a 7 transmembrane domain integral membrane protein consistent with its putative role as an intracellular GPCR.
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Authors | Michio Sone, Seth J Orlow |
Journal | Experimental eye research
(Exp Eye Res)
Vol. 85
Issue 6
Pg. 806-16
(Dec 2007)
ISSN: 0014-4835 [Print] England |
PMID | 17920058
(Publication Type: Journal Article, Research Support, N.I.H., Extramural)
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Chemical References |
- DNA, Complementary
- Eye Proteins
- GPR143 protein, human
- Hemagglutinins
- Membrane Glycoproteins
- Receptors, G-Protein-Coupled
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Topics |
- Albinism, Ocular
(genetics)
- Amino Acid Sequence
- Animals
- COS Cells
- Cell Membrane Permeability
- Chlorocebus aethiops
- DNA, Complementary
(genetics)
- Eye Proteins
(genetics, metabolism)
- Hemagglutinins
- Hydrophobic and Hydrophilic Interactions
- Membrane Glycoproteins
(genetics, metabolism)
- Mutagenesis, Site-Directed
- Receptors, G-Protein-Coupled
(metabolism)
- Sequence Homology, Amino Acid
- Transfection
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