Publication Date

2016-08-01

Availability

Open access

Embargo Period

2016-08-01

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2016-07-15

First Committee Member

Vladlen Slepak

Second Committee Member

Sandra Lemmon

Third Committee Member

Grace Zhai

Fourth Committee Member

Barry Hudson

Abstract

G-protein coupled receptors (GPCRs) mediate the transmission of extracellular signals to generate specific intracellular responses that are crucial for most physiological processes. Timely inactivation of GPCR signal is facilitated by regulators of G-protein signaling (RGS) proteins. This dissertation was focused on the R7 subfamily of RGS proteins. The R7 proteins which include RGS6, RGS7, RGS9 and RGS11 are multi-domain proteins consisting of the RGS, GGL (Gγ-like), DEP (originally identified in Disheveled, EGL-10, and Pleckstrin) and DHEX (DEP helical extension) domains. This subfamily exists as obligate dimers with the atypical Gβ subunit, Gβ5. These complexes are highly expressed in neurons and to a less extent in glands, neuroendocrine cells and in cardiomyocytes where they regulate vision, nociception, motor coordination, learning, reward behavior, heart rate and redox homeostasis. Most of the known functions of R7 proteins are associated with their ability to regulate GPCR signaling. Consistent with this notion, a significant fraction of neuronal R7 proteins is bound to plasma membrane. Plasma membrane localization involves direct interaction with their membrane anchors: R7BP (RGS7 binding protein), R9AP (RGS9 anchoring protein), and the orphan GPCRs: GPR158, and GPR179. Evidence from cell fractionation studies suggests that RGS6, RGS7, and RGS11 distribute about 1:1 between the membrane and soluble cytosolic fractions. The function of cytoplasmic R7 proteins is yet to be unraveled. In DRG neurons, RGS7 localizes to the plasma membrane and cytoplasmic granules. Interestingly, in cell lines, both endogenous and overexpressed R7 proteins showed granular or diffuse cytoplasmic staining with no plasma membrane staining observed. We found that upon overexpression of R7BP and GPR158 in these cell lines, Gβ5/RGS7 complex was recruited to the plasma membrane. This finding suggested that the interaction between RGS7 and its membrane anchors were more stable than the rather dynamic interaction with the granules. We investigated granular RGS7 complexes using in situ chemical crosslinking, which stabilizes the interaction of RGS7 and its binding partners. We found that in mouse brain and transfected cells, cross-linking causes the formation of distinct RGS7 complexes. As the result of the subsequent investigation, we found that Gβ5/RGS7 could form homo-oligomers, possibly (Gβ5-RGS7)3. We also showed that RGS7-RGS7 interaction required the DEP domain, but not the RGS, DHEX domains or the Gβ5 subunit. We began the exploration of the effect of binding partners and G-protein activation on the formation of oligomeric RGS7. Using transfected cells and brain tissue from knockout mice, we demonstrated that R7BP had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of a GTPase-deficient mutant of Gαo was able to prevent RGS7-RGS7 interaction. In contrast, wild-type Gαo had no effect on cross-linking and RGS7-RGS7 co-precipitation. These results suggest that Gαo can prevent RGS7 oligomerization in an activity-dependent manner. We also showed that RGS7 could differentially interact with other R7 RGS proteins. The interaction was strongest with RGS6, which shares 87% similarity with RGS7, and a weak interaction was observed with the more distant RGS9. These results reveal the existence of RGS protein homo- and hetero-oligomers and show regulation of their assembly by R7 RGS binding partners.

Keywords

G protein-coupled receptors (GPCRs), Regulator of G-protein signaling 7 (RGS7), R7-binding protein (R7BP), G protein, oligomer, subcellular localization, protein cross-linking, immunoprecipitation

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