RGS14 is a biochemically diverse protein which integrates G proteins and the MAPKinase signaling cascade Open Access

Cowan, Donald P (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/8910jv31x?locale=en


RGS14 is a biochemically diverse protein which integrates G proteins and the MAPKinase Signaling Cascade

Heterotrimeric G-proteins link extracellular neurotransmitter and hormone receptors to intracellular signaling cascades. In conventional models of G-protein signaling, GPCR's act as guanine nucleotide exchange factors (GEFs) to activate signaling events, and a family of proteins called the regulators of G-protein coupled signaling (RGS) function to negatively modulate G-protein signaling to effectively shut down G-protein signaling cascades. Of particular interest is RGS14, a complex RGS protein that contains a conserved RGS domain, tandem Ras/Rap binding domains (RBD) and a GoLoco/GPR (GL) motif. RGS14 is known to bind activated GTP-bound Gαi and Gio at its RGS domain, Rap2a at its RBD domains, and inactivated GDP-bound Gαi1/3 at its GL domain. Recent data in our lab has shown RGS14 also binds H-Ras, Raf kinases and Ric8a, a known cytosolic GEF. These new data link RGS14 to the MAPKinase cell proliferation pathway and suggest that RGS14 is acting as a novel protein scaffold to regulate local Gα and Raf kinase activity in a non-receptor mediated manner. Early work in our lab has shown RGS14 has the capacity to bind Gαi and activated H-Ras at the same time, but not Gαi and Raf-1 at the same time, suggesting that RGS14 may be acting as a protein switch to regulate Raf kinase activity inside the cell. My data shows that purified RGS14 inhibits Raf-1 mediated phosphorylation of MEK-1/2 in vitro. Additionally, 0ur lab has also previously shown phosphorylation of RGS14 to plays a role in the protein's function. My work has shown RGS14 to be phosphorylated at an unknown site when recruited to the plasma membrane by Gαi. My data suggests the unknown phosphorylated site is likely within the first 213 amino acids of RGS14, or the required binding site for the kinase lies within this region. These findings elucidate the diverse biochemical roles for and potential mechanisms of RGS14 in non-receptor G protein signaling cascades.

Table of Contents

CHAPTER I: Introduction

1.1 G-protein coupled receptors (GPCRs)...2
1.2 Clinical relevance of GPCRs...3
1.3 Conventional GPCR signaling cascades and G proteins...4
1.4 Regulators of G-protein Signaling (RGS) proteins...4

1.4.1 The functional domains and families of RGS proteins
1.4.2 RGS proteins as drug targets

1.5 RGS14...12

1.5.1 Unconventional G protein signaling and RGS14
1.5.2 RGS14 and the MAPkinase signaling cascade
1.5.3 RGS14 is regulated via phosphorylation

1.6 Overall Hypothesis...18

CHAPTER II: RGS14 integrates G proteins and the MAPkinase cascade

2.1 Introduction...23
2.2 Experimental Procedures...25
2.3 Results...28
2.4 Discussion...34

CHAPTER III: RGS14 regulation by phosphorylation

3.1 Introduction...37
3.2 Experimental Procedures...39
3.3 Results...44
3.4 Discussion...61

Chapter IV: Conclusion...65

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