Olfactory Receptor Proteins

Abstract

A rat olfactory epithelium cDNA library was screened for olfactory receptor clones. One of the positively hybridizing cDNA clones was sequenced and found to encode a new member of the olfactory receptor superfamily. This cDNA, termed olp4, was used as a model of olfactory receptor for expression, both in vitro and in vivo. Expression of olp4, as well as of another previously cloned olfactory receptor (F5), was monitored by immunoprecipitation with a monoclonal antibody directed against a Flag peptide epitope tag, inserted at the N-terminus of the open reading frame, and a specific polyclonal antibody against a C-terminal peptide of olp4. Translation in v i m , followed by immunoprecipitation, showed a major olp4-specific band of 27-29 kDa. The olp4 and F5 polypeptides were found to be inserted into microsomal membranes as expected for integral membrane proteins. Expression in vivo of Flag-olp4 in Sf9 insect cells, using the baculovirus expression system, showed a specific polypeptide of the same size as the in vitro species, with an additional band of 34 kDa, which is most likely a glycosylated form. Fluorescence cytometry and immunohistochemical assays demonstrated the localization of the Flag-olp4 product on the cell surface of the infected host Sf9 cells, with the N-terminus and C-terminus in the proper orientation. Affinity chromatography was used for the partial purification of the olp4 polypeptide from infected Sf9 cells. The identification and purification of this expressed olfactory receptor polypeptide could open the way for further characterization and functional studies of the olfactory receptor superfamily members.The superfamily of olfactory receptor proteins [l, 21 is likely to underlie the detection of odorant ligands, thus constituting the molecular basis of the sense of smell. Olfactory receptors are seven-transmembrane-domain receptors, as suggested earlier on the basis of considerable evidence that odorant signals are transduced via GTP-binding proteins (Gproteins) on the cilia of olfactory sensory neurons to activate either the CAMP or the phosphoinositide cascades [3-71. Different olfactory receptors are believed to recognize different odorants, and odor coding is afforded by the specific expression of one or a few olfactory receptor types in each sensory neuron [ 8 -151.The first members of the olfactory receptor gene superfamily were cloned from rat and shown to encode several subfamilies [l]. Later, nearly 100 additional olfactory receptor genes were identified in several species [ l l , 16-19]. A systematic sequence classification by standard methods [20] shows a minimum of eight olfactory receptor families, each constituting one or more subfamilies, leading to the notion that the olfactory receptor gene repertoire is a multigene su- perfamily. Members of this superfamily share at least 35% amino acid sequence identity, as well as several structural features and sequence motifs that clearly distinguish them from the other seven-transmembrane-domain receptors [2]. Many of the olfactory re...

Overexpression, Solubization and Purification of Rat and Human Olfactory Receptors

“…Rabbit polyclonal antibodies were raised against the C-terminal part of rat olp4 ( Table 1). A Flag-epitope tag is also present in the N-terminal part of the recombinant olp4 protein and this peptide is detected by mouse anti-Flag mAb M2 [13]. The olfactory receptor is depicted in the membrane with the transmembrane domains indicated by latin numerals.…”

“…Peptides and antibodies used for the detection of expressed olfactory receptors. olp4, rat olfactory receptor [13] ; OR17-4, human olfactory receptor [27] ; aCT-r4 aCT-h4, rabbit polyclonal antibodies; M2, mouse mAb.…”

“…The full olp4 ORF that contained a Flag-epitope-encoding sequence at its 5' end was amplified by PCR from the plasmid pcDNAI-Flag-olp4 [13]. The sense primer, Flag-Eco-5', contained an EcoRI site (underlined), TA-GAATTCGCCACCATGGATTACAAGGT, and the antisense primer, olp4-C, contained a BamHI site (underlined), TA-ATCCTTAACTCCTGACTGAG.…”

“…Two olfactory receptor genes, rat olp4 [13] and human OR 17-4 [16, 271 were subcloned into the baculovirus transfer vector pAcSGHisNT to create constructs that encoded olfactory receptors with amino-terminal His, tags (Fig. 1).…”

“…The molecular mass and cell-surface disposition of the expressed olp4 protein was studied after immunoprecipitation with specific antibodies. The olp4 protein was shown to reside in the plasma membrane in the correct orientation by means of immunofluorescence and fluorescence-activated-cell-sorting analysis after staining with antibodies against both termini of olp4 [13]. Here we report the overexpression, detergent solubilization and twostep purification of two olfactory receptor proteins : olp4 from rat and OR17-4 from human.…”