ABSTRACT
Regulator of G protein signaling (RGS) proteins negatively regulate receptor-mediated second messenger responses by enhancing the GTPase activity of Galpha subunits. We describe a receptor-specific role for an RGS protein at the level of an individual brain neuron. RGS9-2 and Gbeta(5) mRNA and protein complexes were detected in striatal cholinergic and gamma-aminobutyric acidergic neurons. Dialysis of cholinergic neurons with RGS9 constructs enhanced basal Ca(2+) channel currents and reduced D(2) dopamine receptor modulation of Cav2.2 channels. These constructs did not alter M(2) muscarinic receptor modulation of Cav2.2 currents in the same neuron. The noncatalytic DEP-GGL domain of RGS9 antagonized endogenous RGS9-2 activity, enhancing D(2) receptor modulation of Ca(2+) currents. In vitro, RGS9 constructs accelerated GTPase activity, in agreement with electrophysiological measurements, and did so more effectively at Go than Gi. These results implicate RGS9-2 as a specific regulator of dopamine receptor-mediated signaling in the striatum and identify a role for GAP activity modulation by the DEP-GGL domain.
Subject(s)
Calcium Channels/drug effects , Corpus Striatum/drug effects , RGS Proteins/pharmacology , Receptors, Dopamine D2/drug effects , Animals , Base Sequence , Calcium Channels/metabolism , Cholinergic Fibers/drug effects , Cholinergic Fibers/metabolism , Corpus Striatum/metabolism , DNA/genetics , GTP-Binding Protein beta Subunits/genetics , GTP-Binding Protein beta Subunits/metabolism , In Vitro Techniques , Interneurons/drug effects , Interneurons/metabolism , Protein Structure, Tertiary , RGS Proteins/chemistry , RGS Proteins/genetics , RGS Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Receptors, Dopamine D2/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Signal Transduction/drug effectsABSTRACT
In multicellular organisms from Caenorhabditis elegans to Homo sapiens, the maintenance of homeostasis is dependent on the continual flow and processing of information through a complex network of cells. Moreover, in order for the organism to respond to an ever-changing environment, intercellular signals must be transduced, amplified, and ultimately converted to the appropriate physiological response. The resolution of the molecular events underlying signal response and integration forms the basis of the signal transduction field of research. An evolutionarily highly conserved group of molecules known as heterotrimeric guanine nucleotide-binding proteins (G proteins) are key determinants of the specificity and temporal characteristics of many signaling processes and are the topic of this review. Numerous hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli exert their effects on cells by binding to heptahelical membrane receptors coupled to heterotrimeric G proteins. These highly specialized transducers can modulate the activity of multiple signaling pathways leading to diverse biological responses. In vivo, specific combinations of G alpha- and G beta gamma-subunits are likely required for connecting individual receptors to signaling pathways. The structural determinants of receptor-G protein-effector specificity are not completely understood and, in addition to involving interaction domains of these primary acting proteins, also require the participation of scaffolding and regulatory proteins.
Subject(s)
GTP-Binding Proteins/chemistry , GTP-Binding Proteins/physiology , Animals , Humans , Lipid Metabolism , Molecular Structure , Phosphorylation , Proteins/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction , Structure-Activity RelationshipABSTRACT
The molecular basis of selectivity in G-protein receptor coupling has been explored by comparing the abilities of G-protein heterotrimers containing chimeric Galpha subunits, comprised of various regions of Gi1alpha, Gtalpha, and Gqalpha, to stabilize the high affinity agonist binding state of serotonin, adenosine, and muscarinic receptors. The data indicate that multiple and distinct determinants of selectivity exist for individual receptors. While the A1 adenosine receptor does not distinguish between Gi1alpha and Gtalpha sequences, the 5-HT1A and 5-HT1B serotonin and M2 muscarinic receptors can couple with Gi1 but not Gt. It is possible to distinguish domains that eliminate coupling and are defined as "critical," from those that impair coupling and are defined as "important." Domains within the N terminus, alpha4-helix, and alpha4-helix-alpha4/beta6-loop of Gi1alpha are involved in 5-HT and M2 receptor interactions. Chimeric Gi1alpha/Gqalpha subunits verify the critical role of the Galpha C terminus in receptor coupling, however, the individual receptors differ in the C-terminal amino acids required for coupling. Furthermore, the EC50 for interactions with Gi1 differ among the individual receptors. These results suggest that coupling selectivity ultimately involves subtle and cooperative interactions among various domains on both the G-protein and the associated receptor as well as the G-protein concentration.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gq-G11/chemistry , Transducin/chemistry , Amino Acid Sequence , Animals , Cell Line , Cell Membrane/metabolism , Dimerization , Dose-Response Relationship, Drug , Insecta , Models, Molecular , Molecular Sequence Data , Mutation , Point Mutation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Radioligand Assay , Sequence Homology, Amino AcidABSTRACT
In conclusion, by taking advantage of the overall sequence homology and structural similarity of G alpha subunits, functional chimeric G alpha subunits can be generated and used as tools for the identification of sequence-specific factors that mediate receptor: G protein specificity. The [35S]GTP gamma S binding assay and the affinity shift activity assay are two sensitive biochemical approaches that can be used to assess receptor: G protein coupling in vitro. These in vitro assays limit confounding influences from cellular proteins and allow for the strict control of receptor: G protein ratios.
