10] Use of antisense expression plasmids to attenuate G-protein expression in primary neurons

Abstract

Unraveling signaling pathways in primary neurons has been a central issue for neurobiologists and pharmacologists, but the analysis has been hampered by the paucity of specific molecular tools with which to dissect signal transduction cascades. The advent of gene cloning demonstrated a degree of molecular diversity unknown, and largely unsuspected, in most gene families. This was nowhere more apparent than in G protein–coupled receptor (GPCR)-mediated signal transduction. This signaling cascade starts with the activation of a GPCR and consequent catalytic guanosine diphosphate/guanosine triphosphate (GDP/GTP) exchange on the α subunit of a trimeric G protein, thereby causing dissociation into a GTP-bound α subunit and a βγ dimer, either of which can act on a myriad of channels or enzymes. Currently, genetic diversity accounts for more than 1,000 GPCRs, 17 G α subunits, 5 G β subunits, and 12 G γ subunits, as well as hundreds of ion channel and enzyme effectors. This diversity is further increased by the existence of splice variants. The fact that the recognition of this molecular diversity largely relied on molecular cloning bears testament to the degree of similarity among the gene products, which was not discernible using conventional biochemical or pharmacological agents. This similarity offers a challenge to develop specific molecular tools, because the degree of amino acid identity among cognate members of a gene family can be so great as to preclude the design of agents that will readily distinguish individual relatives.