Dopamine acts through two classes of G protein-coupled receptor (D1-like and D2-like) to modulate neuron activity in the brain. While subtypes of D1-and D2-like receptors are coexpressed in many neurons of the mammalian brain, it is unclear how signaling by these coexpressed receptors interacts to modulate the activity of the neuron in which they are expressed. D1-and D2-like dopamine receptors are also coexpressed in the cholinergic ventral-cord motor neurons of Caenorhabditis elegans. To begin to understand how coexpressed dopamine receptors interact to modulate neuron activity, we performed a genetic screen in C. elegans and isolated mutants defective in dopamine response. These mutants were also defective in behaviors mediated by endogenous dopamine signaling, including basal slowing and swimming-induced paralysis. We used transgene rescue experiments to show that defects in these dopamine-specific behaviors were caused by abnormal signaling in the cholinergic motor neurons. To investigate the interaction between the D1-and D2-like receptors specifically in these cholinergic motor neurons, we measured the sensitivity of dopamine-signaling mutants and transgenic animals to the acetylcholinesterase inhibitor aldicarb. We found that D2 signaling inhibited acetylcholine release from the cholinergic motor neurons while D1 signaling stimulated release from these same cells. Thus, coexpressed D1-and D2-like dopamine receptors act antagonistically in vivo to modulate acetylcholine release from the cholinergic motor neurons of C. elegans.
The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.Four universally conserved GTPases interact with the ribosome to coordinate the initiation, elongation, and termination of protein synthesis. Initiation factor 2/eukaryotic translation initiation factor 5B (IF2/eIF5B) initially binds to the small ribosomal subunit and promotes subunit joining during translation initiation (5,30,36). The factor EF-Tu in bacteria or eEF1A in eukaryotes delivers aminoacyl-tRNAs to the A site of the ribosome, while the bacterial factor EF-G, or eEF2 in eukaryotes, promotes translocation of peptidyl-tRNA from the A to the P site during translation elongation (reviewed in references 1, 37, and 38). Finally, the factor RF3 (or eRF3 in eukaryotes) functions with the stop codon recognition factors to promote translation termination (25). As expected, the conserved GTP binding (G) domains of these factors contain the sequence motifs that are hallmarks of all G proteins. In addition to the Walker A-box GXXXGK(T/S) motif (G-1), which interacts with ␣ and  phosphates of GTP, and the G-4 motif NKXD, which specifies guanine nucleotide recognition, the G-3 motif DXXG within the mobile switch II element is also conserved (48). Finally, X-ray structure analyses revealed that in addition to the G domain, the four translation GTPases share a conserved -barrel domain II (28, 40). Binding of GTP versus GDP to EF-Tu altered the position of the switch II element and induced a significant rearrangement of domain II relative to the G domain (9, 26). Likewise, GTP binding to eIF5B induced movement of switch II and a rotation-like movement of domain II (39). As these gross structural rearrangements alter the aminoacyl-tRNA and/or ribosome binding affinities of the factors, it can be proposed that GTP switches govern the interaction of the translational GTPases with the ribosome.Cryo-electron microscopy (cryo-EM) studies have revealed the binding sites for EF-Tu, EF-G, IF2, and RF3 on the ribosome (2,3,20,27,33,47,49,55). All four factors were found to bind in the ribosomal entry site, the cleft on th...
Translation initiation factor eIF5B promotes GTP-dependent ribosomal subunit joining in the final step of the translation initiation pathway. The protein resembles a chalice with the α-helix H12 forming the stem connecting the GTP-binding domain cup to the domain IV base. Helix H12 has been proposed to function as a rigid lever arm governing domain IV movements in response to nucleotide binding and as a molecular ruler fixing the distance between domain IV and the G domain of the factor. To investigate its function, helix H12 was lengthened or shortened by one or two turns. In addition, six consecutive residues in the helix were substituted by Gly to alter the helical rigidity. Whereas the mutations had minimal impacts on the factor's binding to the ribosome and its GTP binding and hydrolysis activities, shortening the helix by six residues impaired the rate of subunit joining in vitro and both this mutation and the Gly substitution mutation lowered the yield of Met-tRNA(i)(Met) bound to 80S complexes formed in the presence of nonhydrolyzable GTP. Thus, these two mutations, which impair yeast cell growth and enhance ribosome leaky scanning in vivo, impair the rate of formation and stability of the 80S product of subunit joining. These data support the notion that helix H12 functions as a ruler connecting the GTPase center of the ribosome to the P site where Met-tRNA(i)(Met) is bound and that helix H12 rigidity is required to stabilize Met-tRNA(i)(Met) binding.
Dopamine signaling modulates voluntary movement and reward-driven behaviors by acting through G protein-coupled receptors in striatal neurons, and defects in dopamine signaling underlie Parkinson's disease and drug addiction. Despite the importance of understanding how dopamine modifies the activity of striatal neurons to control basal ganglia output, the molecular mechanisms that control dopamine signaling remain largely unclear. Dopamine signaling also controls locomotion behavior in Caenorhabditis elegans. To better understand how dopamine acts in the brain we performed a large-scale dsRNA interference screen in C. elegans for genes required for endogenous dopamine signaling and identified six genes (eat-16, rsbp-1, unc-43, flp-1, grk-1, and cat-1) required for dopamine-mediated behavior. We then used a combination of mutant analysis and cell-specific transgenic rescue experiments to investigate the functional interaction between the proteins encoded by two of these genes, eat-16 and rsbp-1, within single cell types and to examine their role in the modulation of dopamine receptor signaling. We found that EAT-16 and RSBP-1 act together to modulate dopamine signaling and that while they are coexpressed with both D1-like and D2-like dopamine receptors, they do not modulate D2 receptor signaling. Instead, EAT-16 and RSBP-1 act together to selectively inhibit D1 dopamine receptor signaling in cholinergic motor neurons to modulate locomotion behavior.
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