Ric-8B, an olfactory putative GTP exchange factor, amplifies signal transduction through the olfactory-specific G-protein Galphaolf

Abstract

The olfactory system is able to detect a large number of chemical structures with a remarkable sensitivity and specificity. Odorants are first detected by odorant receptors present in the cilia of olfactory neurons. The activated receptors couple to an olfactory-specific G-protein (Golf), which activates adenylyl cyclase III to produce cAMP. Increased cAMP levels activate cyclic nucleotide-gated channels, causing cell membrane depolarization. Here we used yeast two-hybrid to search for potential regulators for Galphaolf. We found that Ric-8B (for resistant to inhibitors of cholinesterase), a putative GTP exchange factor, is able to interact with Galphaolf. Like Galphaolf, Ric-8B is predominantly expressed in the mature olfactory sensory neurons and also in a few regions in the brain. The highly restricted and colocalized expression patterns of Ric-8B and Galphaolf strongly indicate that Ric-8B is a functional partner for Galphaolf. Finally, we show that Ric-8B is able to potentiate Galphaolf-dependent cAMP accumulation in human embryonic kidney 293 cells and therefore may be an important component for odorant signal transduction.

Figure 1.

Ric-8B interacts with Gαolf. a, Amino acid sequence alignment of mouse Ric-8B and Ric-8A. Shaded sequences indicate areas of amino acid identity. The nine synembryn signatures are underlined in gray. The region corresponding to exon 9 is underlined in black. b, Schematic representation of the Ric-8B gene structure. Exons are represented by black boxes, and introns are represented by thick lines. The initiator methionine ATG codon is in exon 1, and the stop codon is in the beginning of exon 10. The Ric-8B cDNA contains all of the 10 exons. The alternative spliced variant (denominated Ric-8BΔ9) also identified in the two-hybrid screen does not contain exon 9, as indicated. c, Interaction between Ric-8B and Gαolf. Bait strains expressing full-length Gαolf (amino acids 1-381) or different regions of Gαolf (amino acids 1-42, 72-188, or 352-381) were mated with target strains expressing full-length Ric-8B or Ric-8BΔ9. X-gal was used to score positive interactions. pBait (B), which constitutively expresses a LexA bait fusion protein that interacts with the fusion protein from pTarget (T), was used as positive control.

Figure 5.

Amplification of cAMP accumulation by Ric-8B. a, Production of cAMP was measured in HEK293 cells transfected as indicated with Gαolf, Ric-8B, Ric-8BΔ9, Gα_s, and β₂AR expression vectors. Activation was recorded in the absence (white bars) or presence (black bars) of 10 μm isoproterenol. The data are expressed as the means ± SE from three different experiments, each performed in duplicate. cAMP accumulation is presented as picomoles per million cells. - indicates untransfected cells (control). b, Same as in a, except that cells were transfected with D₁R instead of β₂AR and the agonist was dopamine (10 μm). c, Cells were cotransfected with Gαolf and D₁R (white bars) or with Gαolf, D₁R, and Ric-8B (black bars). Production of cAMP was measured after dopamine stimulation for the indicated periods of time. - indicates that no dopamine was added. The data are expressed as the means of two independent experiments.

Figure 2.

Ric-8B is mainly expressed in the olfactory epithelium. a, Northern blot analysis of mouse total RNA: 1, olfactory epithelium; 2, whole eye; 3, brain; 4, skeletal muscle; 5, heart; 6, kidney; 7, liver; 8, lung; 9, testis; 10, thymus. A probe prepared from Ric-8B was hybridized using stringent conditions. Hybridization with GAPDH probe indicated that similar amounts of RNA were loaded in each lane. Sizes are given in kilobases. b, RT-PCR was conducted to amplify Ric-8B, Gαolf, and GAPDH from the same tissues as in a. (-), Negative control (no cDNA was added to the reaction). c, Semiquantitative RT-PCR was conducted to amplify Ric-8B from olfactory epithelium RNA after 20, 25, 30, and 35 cycles, as indicated. The PCR product sizes expected for Ric-8B using the primers that flank the ninth exon are 462 bp (Ric-8B) and 342 bp (Ric-8BΔ9). M, Molecular weights are given in kilobases.

Figure 3.

Distribution of Ric-8B mRNA in the olfactory system. a-f, Coronal sections through the nasal cavity were hybridized with antisense digoxigenin-labeled probes specific for Ric-8B (a), Gαolf (b), and Gα_s (c). Corresponding sense digoxigenin-labeled probes were used as negative controls (d-f). Neurons in the olfactory epithelium that hybridize with the Ric-8B probe show a pattern typical of that seen with the Gαolf probe. g-i, Magnified regions from a-c showing the distribution of stained cells within the olfactory epithelium. Ric-8B (g) and Gαolf (h) probes hybridize to mature neurons (ON) but not to the most apical layer of cells closest to the lumen [supporting cells (SC)] or to the immature neurons localized in the basal layer of the epithelium (BC). The Gαs probe (i), conversely, hybridizes preferentially to immature cells localized in the basal layer of the epithelium. j-l, Sections cut through the olfactory and vomeronasal epithelia. Ric-8B (j) and Gαolf (k) probes hybridize to neurons in the olfactory epithelium but not to vomeronasal neurons. OMP (l) is expressed in the olfactory and vomeronasal epithelia.

Figure 4.

Comparison of mRNA expression patterns for Ric-8B, Gαolf, and Gα_s in the adult mouse brain. a-f, Sagittal brain sections were hybridized with antisense digoxigenin-labeled probes specific for Ric-8B (a), Gαolf (c), and Gα_s (e). Corresponding sense digoxigenin-labeled probes were used as negative controls (b, d, f). g-i, Coronal brain sections were hybridized with antisense digoxigenin-labeled probes specific for Ric-8B (g), Gαolf (h), and Gα_s (i). Ric-8B is expressed in regions known to express high levels of Gαolf and that show little or no expression of Gα_s: the striatum (St), nucleus accumbens (Na), and olfactory tubercle (Ot). Ric-8B expression was also detected in the piriform cortex (Pc), dentate gyrus (Dg), and CA3 region of the hippocampus, in which both Gαolf and Gα_s are also expressed. Ce, Cerebellum.