Co-localization of odorant receptors expressed in lepidopteran insect cells.

Abstract

<p>(<b>A</b>) Expression constructs for N-terminally (mycOR1, mycOR2) or C-terminally tagged receptors (OR1myc) were transfected in Bm5 cells (in the absence/presence of OR7) and the localization of the expressed ORs was detected using anti-Myc antibody. Control indicates transfection with empty expression vector. (<b>B</b>) Co-localization of OR2 with the plasma membrane marker wheat germ agglutinin. Cells expressing OR2myc were double stained with WGA-Texas Red-X conjugate (b, e, f) and with anti-myc antibody (a, d, g) in the presence or absence of saponin (a-f and g-i, respectively). (<b>C</b>) Detection of ORs in the membrane fraction of stable cell lines coexpressing mycOR1 (lane 2) or mycOR2 (lane 3) along with flagOR7. Immunoblotting was performed with anti-Myc and anti-Flag antibodies (upper and lower panels, respectively). Membranes from Bm5 untransfected cells were used as a negative control (lane 1). (<b>D</b>) Co-localization of OR1 or OR2 with OR7. Bm5 cells were co-transfected with expression plasmids for N-terminally tagged mycOR1 or mycOR2 together with N-terminally tagged flagOR7 expression vector. Tagged ORs were detected with anti-Myc/anti-mouse fluorescein-labelled IgG and anti-Flag/anti-rabbit Alexa fluor-labelled IgG as indicated and counter-stained with DAPI. (<b>E</b>) Pull-down assays showing heteromerization between OR1 and OR7 or OR2 and OR7. Extracts containing C-terminally Myc-His-tagged OR7 were incubated with Ni²⁺-NTA beads and bound protein complexes were analyzed by Western blot by anti-Flag antibody (upper panel) for the presence of N-terminally Flag-tagged OR1 and OR2 or by anti-Myc antibody (lower panel) to detect OR7mychis.</p