The N‐terminal acidic residue of the cytosolic helix 8 of an odorant receptor is responsible for different response dynamics via G‐protein

Abstract

Interaction Homology model a b s t r a c tWe previously observed highly rapid and robust response of murine olfactory receptor S6 (mOR-S6) with chimeric Ga 15_olf , compared to Ga 15 . To identify residues responsible for this difference in response, mutations of the cytosolic helix 8 were analyzed in a heterologous functional expression system. The N-terminal hydrophobic core between helix 8 and TM1-2 of mOR-S6 is important for activation of both Ga 15_olf and Ga 15 . Point mutation of a helix 8 N-terminal acidic residue eliminated the differences in response dynamics via Ga. This result suggests that an N-terminal acidic residue of helix 8 is responsible for rapid response via Ga 15_olf .

Functional Role of the C-Terminal Amphipathic Helix 8 of Olfactory Receptors and Other G Protein-Coupled Receptors

“…In each step, a specific interaction with a higher binding affinity of a ligand for a receptor is of higher priority. Evidence suggests that initial transient and specific interactions facilitate the shared, extensive (more stable) and partially type-specific interactions [4,5,7], but not vice versa (i.e., shared interactions do not facilitate specific interactions in our model).…”

“…This avoids functional disorders resulting from stimulation or inactivation of non-target effector proteins. This functional model allows us to predict intra- and inter-molecular interactions, as follows: An agonist molecule binds to a specific binding site of a target GPCR (first semi-activation).Binding of a specific agonist induces the structural rearrangement of GPCR transmembrane domains, leading to conformational changes and transition to an active state (activation by an agonist).The activated GPCR initially and transiently interacts with a target heterotrimeric G protein [7] comprised of α-, β- and γ-subunits (second semi-activation).In the initial and transient interaction between the GPCR and a semi-activated target G protein, displacement of helix-α5 of the Gα subunit towards TM3 of the GPCR facilitates the formation of a more stable, ternary activated GPCR–heterotrimeric G protein complex that is mediated by shared and/or partially specific molecular interactions [5] (full activation).In the stable, ternary activated GPCR–heterotrimeric G protein complex, the Gα subunit releases GDP from the binding pocket.A GTP then binds to the nucleotide-free Gα subunit, followed by dissociation of Gα and βγ subunits from the GPCR [5]. …”

“…The activated GPCR initially and transiently interacts with a target heterotrimeric G protein [7] comprised of α-, β- and γ-subunits (second semi-activation).…”

“…In rhodopsin (Rhod), helix 8 acts as a membrane surface recognition domain, and adopts a helical structure only in the presence of membranes or membrane mimetics [9]. Helix 8 begins after a short linker following TM7, at the end of which the conserved NPxxY motif is located, as shown in a selection of class A GPCR sequences (Figure 1) [3,4,5,6,7]. The short linker between TM7 and helix 8 is also important, as described in Section 5 below.…”

“…Recently, we found that the second residue of the amphipathic helix 8 in the C-terminal domain of the murine olfactory receptor S6 ( m OR-S6), a GPCR superfamily member, is responsible for initial transient and specific interactions with chimeric Gα 15_olf , but not with Gα 15 [7]. Our mutagenesis analysis also indicates that the hydrophobic core that is buried between the amphipathic helix 8 and transmembrane domains 1–2 (TM1–2) of m OR-S6 are important for activation of both Gα 15_olf and Gα 15 .…”