Olfactory receptor surface expression is driven by association with the β₂-adrenergic receptor

Abstract

Olfactory receptors (ORs) comprise more than half of the large class I G protein-coupled receptor (GPCR) superfamily. Although cloned over a decade ago, little is known about their properties because wild-type ORs do not efficiently reach the cell surface following heterologous expression. Receptor-receptor interactions strongly influence surface trafficking of other GPCRs, and we examined whether a similar mechanism might be involved in OR surface expression. Olfactory neurons are known to express ␤-adrenergic receptors (ARs), and we found that coexpression with ␤ 2 -ARs, but not any other AR subtypes, dramatically increased mouse 71 (M71) OR surface expression in human embryonic kidney 293 cells. A persistent physical interaction between M71 ORs and ␤2-ARs was shown by coimmunoprecipitation and by cointernalization of the two receptors in response to their specific ligands. Also, coexpression of wild-type M71 ORs with ␤2-ARs resulted in cAMP responses to the M71 ligand acetophenone. Finally, in situ hybridization studies showed extensive colocalization of M71 OR and ␤2-AR expression in mouse olfactory epithelium. These data demonstrate the successful heterologous surface expression of a functional wild-type OR and reveal that persistent physical association with other GPCRs can control OR surface expression. P erception of smell begins with stimulation of olfactory receptors (ORs) on neurons within the olfactory epithelium, leading to excitation and propagation of currents to the main olfactory bulb (1, 2). ORs are class I G protein-coupled receptors (GPCRs) that signal through stimulation of G␣ olf , which leads to activation of type III adenylyl cyclase and opening of cAMPgated cation channels (3). Since the completion of the human and mouse genome sequencing projects, Ϸ350 receptors in humans (4) and Ϸ1,000 receptors in mice (5) have been identified, presumably to aid in the selective recognition of Ͼ100,000 different odors. However, the mechanism by which the olfactory system selectively recognizes specific odors remains unclear. It was initially hypothesized that each olfactory neuron expresses a single OR and that the axons of olfactory neurons expressing the same OR then converge in the main olfactory bulb (6, 7). However, increasing evidence suggests that detection is substantially more complex than previously thought. For example, olfactory neurons are not restricted to expression of a single OR subtype (8). In addition to ORs, olfactory neurons can express many other receptors, which facilitate modulation of olfactory responses by hormones and neurotransmitters. For example, epinephrine stimulation of endogenous ␤-adrenergic receptors (ARs) modifies the signaling of coexpressed ORs within olfactory neurons (9). Furthermore, multiple OR subtypes can respond to the same ligand, a single OR can respond to multiple ligands (10-12), and structurally similar odorant ligands can act as either agonists or antagonists (13). Thus, as the complexity of the olfactory system becomes increasingly clear, the need t...

Specificity of Olfactory Receptor Interactions with Other G Protein-coupled Receptors

“…In previous confocal studies, we noted that the ␤ 2 -AR did not appear to enhance the surface localization of two ORs that are distantly related to M71: hOR17-40 and rat I7 (16). Similarly, in the current analysis, co-expression with ␤ 2 -AR, P2Y 1 R, P2Y 2 R, or A 2A R did not significantly alter hOR17-40 or rat I7 plasma membrane expression as assessed in luminometer assays (Fig.…”

“…We previously found that ACP stimulation of wild type M71 (WT-M71) expressed in HEK-293 cells did not result in detectable receptor signaling, consistent with the lack of receptor expressed at the plasma membrane, but stimulation of WT-M71 co-expressed with ␤ 2 -AR did result in significant cAMP generation (16). These studies demonstrated that when in complex with ␤ 2 -AR, heterologously expressed WT-M71 can signal via cAMP generation, as has been reported for many examples of odorant-induced signaling in native OSNs (1,34).…”

“…upon Co-expression with the Purinergic Receptors P2Y 1 R, P2Y 2 R, and A 2A R-We have previously reported that association of the OR M71 with the ␤ 2 -AR alleviates intracellular retention and yields functional M71 localized at the plasma membrane (16). To determine the specificity of such GPCR-OR interactions and identify whether other GPCRs are similarly capable of enhancing M71 plasma membrane localization, we conducted a screen co-expressing M71 with a multitude of other GPCRs.…”

“…To determine the specificity of such GPCR-OR interactions and identify whether other GPCRs are similarly capable of enhancing M71 plasma membrane localization, we conducted a screen co-expressing M71 with a multitude of other GPCRs. These co-expressed GPCRs represent families from which at least one receptor subtype is reportedly expressed in the olfactory epithelium (OE) and/or olfactory bulb (16,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32) and include the trace amine-associated receptors, a new class of chemosensory receptor in the OE (32). M71 tagged at the N terminus with FLAG and at the C terminus with GFP (FLAG-M71-GFP) was expressed alone and in combination with each of the other GPCRs by transient transfection in HEK-293 cells.…”

“…We previously found that association with the ␤ 2 -adrenergic receptor (AR) results in enhanced surface expression and functionality of the OR M71 (16). A natural question of interest following this finding was whether GPCRs other than ␤ 2 -AR are capable of assembling with M71 to promote its surface expression and conversely whether ORs beyond M71 can undergo heterodimerization with nonolfactory GPCRs to enhance their trafficking to the cell surface.…”