Structural determinants for membrane trafficking and G protein selectivity of a mouse olfactory receptor.
The G protein-coupled olfactory receptor (OR) superfamily plays a critical role in recognizing a broad range of odorants. Each OR appears to recognize odorants based on similarities in molecular structures such that mOR-EG, a mouse OR, binds eugenol, vanillin, and some other structurally related odorants. Only a few ORs, however, have been characterized functionally due to the difficulties in expressing ORs in heterologous cells. In this report, we demonstrate roles of the N- and C-terminal domains as key elements in the functional expression and signal transducing activity of an OR. Disruption of the N-terminal glycosylation site of the mOR-EG completely impaired its membrane trafficking to the cell surface. Functional expression of the mOR-EG was greatly enhanced by addition of extra N-terminal glycosylation sequences. Addition of a C-terminal epitope-tag or C-terminal truncation significantly reduced the odorant-response activity, although the receptors were properly targeted to the plasma membrane. Analysis of a series of truncated ORs revealed a region in the C-terminus that was crucial for the receptor activity. Replacement of the C-terminal portion of the mOR-EG with that of rhodopsin disrupted the coupling to G(alphas) but not to G(alpha15), demonstrating that the C-terminus is involved in regulating G protein specificity. These results suggest that glycosylation of the N-terminal portion is critical for OR expression and membrane trafficking, while the C-terminal portion plays a role in defining proper conformation, which, in turn, specifies the G protein selectivity of the OR. This information helps clarify the mechanisms that regulate membrane trafficking and G protein interaction of the OR superfamily.
Katada
Sayako
S
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Japan.
Tanaka
Motonari
M
Touhara
Kazushige
K
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D005054:Eugenol / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D018034:Olfactory Receptor Neurons / Q000187:drug effects / Q000378:metabolism*; D011233:Precipitin Tests / Q000379:methods; D011487:Protein Conformation; D021381:Protein Transport; D018035:Receptors, Odorant / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D025603:Sensory Rhodopsins / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection / Q000379:methods
D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins; D025603:Sensory Rhodopsins; D005054:Eugenol; D002217:Carbachol; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D002118:Calcium