The ocular albinism type 1 gene product, OA1, spans intracellular membranes 7 times

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 Cterminus 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.

Interaction between G Protein-Coupled Receptor 143 and Tyrosinase: Implications for Understanding Ocular Albinism Type 1

“…Thus, we established a cell system to study direct GPR143 and tyrosinase interactions. Since previous studies showed that GPR143 is a 7-transmembrane protein (Sone and Orlow, 2007) and tyrosinase is a single membrane-spanning enzyme, both with lumen facing c-termini, we tagged the C-termini of both generating GPR143-EYFP and TYR-ECFP fusion proteins. Thus the fluorescent proteins are located on the same side of the membrane.…”

“…GPR143 mutations which affect function may compromise downstream signaling, permitting continuous import of melanosomal proteins into melanosomes and sustained melanogenesis resulting in giant organelles. Based on its topological orientation (Schiaffino et al, 1999; Sone and Orlow, 2007), GPR143 ligands should bind in the organelle lumen and transduce information to the cytosol through heterotrimeric-G protein activation. In this way, GPR143 could function as a “sensor” of melanosomal maturation and prevent formation of macro-organelles (Schiaffino and Tacchetti, 2005).…”

Signaling pathways in melanosome biogenesis and pathology

“…Consistently, proteomic analysis of a purified early melanosome fraction obtained from MNT1 cells identified OA1 as a constituent of these organelles (Basrur et al, 2003). Based on sequence homologies and on experimental evidences (Schiaffino and Tacchetti, 2005; Sone and Orlow, 2007), the topological orientation of OA1 on the organelle membrane appears equivalent to canonical seven transmembrane receptors, with the N-terminus toward the lumen and with the C-terminus toward the cytoplasm, and therefore, by comparison with receptors at the plasma membrane, a putative ligand should bind OA1 on the lumenal side of the organelles.…”

Expression of ocular albinism 1 (OA1), 3, 4- dihydroxy- L-phenylalanine (DOPA) receptor, in both neuronal and non-neuronal organs

“…The oa1 mutation leads to giant melanosomes in retinal pigmental epithelium in turn to bring the ocular albinism. OA1 belongs to G-protein-coupled receptor (GPCR) family (Sone and Orlow, 2007). DOPA stimulation on OA1 increases the intracellular calcium concentration via Gq-activation (Lopez et al, 2008).…”