Abstract
The Gα subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-based cloning methods and transcriptome data mining to identity Gα subunits in the western tarnished plant bug (Lygus hesperus Knight). Among the nine sequences identified were single variants of the Gαi, Gαo, Gαs, and Gα12 subfamilies and five alternative splice variants of the Gαq subfamily. Sequence alignment and phylogenetic analyses of the putative L. hesperus Gα subunits support initial classifications and are consistent with established evolutionary relationships. End-point PCR-based profiling of the transcripts indicated head specific expression for LhGαq4, and largely ubiquitous expression, albeit at varying levels, for the other LhGα transcripts. All subfamilies were amplified from L. hesperus chemosensory tissues, suggesting potential roles in olfaction and/or gustation. Immunohistochemical staining of cultured insect cells transiently expressing recombinant His-tagged LhGαi, LhGαs, and LhGαq1 revealed plasma membrane targeting, suggesting the respective sequences encode functional G protein subunits.
Highlights
Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are molecular switches that mediate many extracellular signaling processes by coupling cell surface receptor activation with the diverse signal transduction effector molecules that drive cellular responses
To identify G proteins expressed in L. hesperus (LhG), we initially utilized a homology-based approach with degenerate primers designed to conserved regions of G proteins and both PCR and nested PCR conditions
Further extension of the partial sequences using conventional RACE PCR methods identified putative start and stop codons. Primers designed to those regions facilitated amplification of the respective open reading frames (ORFs)
Summary
Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are molecular switches that mediate many extracellular signaling processes by coupling cell surface receptor activation with the diverse signal transduction effector molecules that drive cellular responses. The intrinsic GTPase activity of G hydrolyzes GTP to GDP, which promotes reassociation of the heterotrimeric G protein complex and terminates the signal [1,2,3] Based on this intermediary molecular role, heterotrimeric G proteins play pivotal roles in determining the specificity and duration of the cellular response to extracellular signals. Other studies though have reported a G protein-coupled metabotropic component in olfactory receptor activation [16] In support of this pathway, G subunits are expressed in chemosensory tissues [17,18,19,20,21],. We performed detailed sequence comparisons of the L. hesperus transcripts with those from other insects, profiled transcript expression levels, and examined the subcellular localization of a subset of recombinantly expressed L. hesperus G proteins in cultured insect cells
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