Abstract
Chemoelectrical signal transduction in olfactory neurons appears to involve intracellular reaction cascades mediated by heterotrimeric GTP-binding proteins. In this study attempts were made to identify the G protein subtype(s) in olfactory cilia that are activated by the primary (odorant) signal. Antibodies directed against the alpha subunits of distinct G protein subtypes interfered specifically with second messenger reponses elicited by defined subsets of odorants; odor-induced cAMP-formation was attenuated by Galphas antibodies, whereas Galphao antibodies blocked odor-induced inositol 1,4, 5-trisphosphate (IP3) formation. Activation-dependent photolabeling of Galpha subunits with [alpha-32P]GTP azidoanilide followed by immunoprecipitation using subtype-specific antibodies enabled identification of particular individual G protein subtypes that were activated upon stimulation of isolated olfactory cilia by chemically distinct odorants. For example odorants that elicited a cAMP response resulted in labeling of a Galphas-like protein, whereas odorants that elicited an IP3 response led to the labeling of a Galphao-like protein. Since odorant-induced IP3 formation was also blocked by Gbeta antibodies, activation of olfactory phospholipase C might be mediated by betagamma subunits of a Go-like G protein. These results indicate that different subsets of odorants selectively trigger distinct reaction cascades and provide evidence for dual transduction pathways in olfactory signaling.
Highlights
Chemoelectrical signal transduction is considered to be mediated via intracellular reaction cascades triggered by G protein-coupled receptors [1]
Antibodies directed against the ␣ subunits of distinct G protein subtypes interfered with second messenger reponses elicited by defined subsets of odorants; odor-induced cAMP-formation was attenuated by G␣s antibodies, whereas G␣o antibodies blocked odor-induced inositol 1,4,5-trisphosphate (IP3) formation
Activation-dependent photolabeling of G␣ subunits with [␣-32P]GTP azidoanilide followed by immunoprecipitation using subtype-specific antibodies enabled identification of particular individual G protein subtypes that were activated upon stimulation of isolated olfactory cilia by chemically distinct odorants
Summary
Chemoelectrical signal transduction is considered to be mediated via intracellular reaction cascades triggered by G protein-coupled receptors [1]. Even an olfactory-specific isoform of Gs (Golf) has been discovered [18] It is currently unclear how many and which type of G proteins are involved in olfactory signal transduction. To approach the question of which G protein subtype(s) may mediate the transduction processes in olfactory sensory cells, it is necessary to identify the G protein that is activated upon stimulation with distinct odor ligands. This can be accomplished by an activationdependent labeling procedure [19], in which receptor-activated G protein ␣ subunits are photolabeled using the hydrolysisresistant GTP-analogue [␣-32P]GTP azidoanilide. The data indicate that cAMP- and IP3-inducing odorants result in labeling of different G protein subtypes
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