Dynamic regulation of R7BP (R7 Binding Protein) containing R7 RGS (R7 Regulators of G protein Signaling) protein complexes: role in controlling neuronal dopamine and opioid signaling in the striatum.

University of Minnesota Ph.D. dissertation. February 2010. Major:Pharmacology. Advisor: Dr. Kirill A. Martemyanov. 1 computer file (PDF); x, 131 pages.G protein-coupled receptor (GPCR) signaling pathways mediate the transmission of signals from the extracellular environment to the generation of cellular responses, a process that is critically important for neurons and neurotransmitter action. The ability to promptly respond to rapidly changing stimulation requires timely inactivation of G proteins, a process controlled by a family of specialized proteins known as regulators of G protein signaling (RGS). The R7 group of RGS proteins (R7 RGS) has received special attention due to their pivotal roles in the regulation of a range of crucial neuronal processes such as vision, motor control, reward behavior and nociception in mammals. One member of the R7 RGS family, RGS9-2 has been previously implicated as an essential modulator of signaling through neuronal dopamine and opioid G protein coupled receptors. RGS9-2 is specifically expressed in striatal neurons where it forms complexes with R7BP (R7 RGS Binding Protein), which we have found to ultimately affect several critical properties of RGS9-2. First, it is this interaction with R7BP which is necessary for determining the subcellular targeting of RGS9-2 to the plasma membrane and to the specialized neuronal compartment of excitatory synapses, the postsynaptic density. Secondly, R7BP plays a selective role amongst the R7 RGS family in determining the proteolytic stability of RGS9-2. Further characterization of R7 RGS complexes in the striatum revealed that two equally abundant R7 RGS proteins, RGS9-2 and RGS7, are unequally coupled to the R7BP subunit which is present in complex predominantly with RGS9-2 rather than with RGS7. However, upon changes in neuronal activity the subunit composition of these complexes in the striatum undergoes rapid and extensive remodeling. Changes in the neuronal excitability or oxygenation status result in extracellular calcium entry, uncoupling RGS9-2 from R7BP, triggering its selective degradation. Concurrently, released R7BP binds to cytoplasmic RGS7 and recruits it to the plasma membrane and the postsynaptic density. These observations introduce activity dependent remodeling of R7 RGS complexes as a new molecular plasticity mechanism in striatal neurons and suggest a general model for achieving rapid posttranslational subunit rearrangement in multi-subunit complexes. The physiological consequence of this remodeling process appears to play a role in determining the signaling sensitivity to dopamine stimulation. Considering that upon the genetic elimination of RGS9, all available R7BP is funneled towards complex formation with RGS7, not only are RGS9 controlled GPCR signaling pathways affected, but those controlled by RGS7 as well. RGS9 knockout mice have an increased sensitivity to dopamine and opioid receptor stimulation and consequently display altered motor and reward behavior. The question arises as to the role of modulation of RGS7 function in controlling these behaviors. Since the function of RGS9-2 is controlled by its association with R7BP, we would predict that the elimination of R7BP would lead to similar alterations in striatal physiology for RGS9 controlled pathways. While at the same time, RGS7 would be largely unaffected by the elimination of R7BP, thus RGS7 controlled pathways would predictably remain unaltered. Using this rationale, we report that elimination of R7BP in mice results in motor coordination deficits and greater locomotor response to morphine administration consistent with the essential role of RGS9 in controlling these behaviors and the critical role played by R7BP in maintaining RGS9-2 expression in the striatum. However, in contrast to previously reported observations with RGS9-2 knockouts, mice lacking R7BP do not exhibit higher sensitivity to locomotor-stimulating effects of cocaine, suggesting a role for RGS7 in controlling dopamine sensitivity. Using a striatum-specific knockdown approach, we demonstrate that the sensitivity of motor stimulation to cocaine is indeed dependent on RGS7 function. These results indicate that dopamine signaling in the striatum is controlled by concerted interplay between two RGS proteins, RGS7 and RGS9-2, which are balanced by a common subunit, R7BP