Human RGS14: Relationship Between Sequence Variation, Brain Distribution, and Protein Function Public
Squires, Katherine (Spring 2018)
Published
Abstract
Regulators of G protein signaling (RGS) proteins have become appreciated for their diverse and defining roles in the regulation of an array of human physiological processes, including those that manifest as both diseases and traits. While our understanding of the roles of RGS proteins in disease propensity and progression is based largely on knockout studies, our understanding of the diversity of genetic variation reflected in the human population, and whether these genetic variants recapitulate knockout mouse models in the lab, has remained largely unexplored. Population genome/exome sequencing has provided a wealth of human genetic variant data, with which we can mine for functionally-relevant variants in RGS proteins. While loss or mutation of some proteins triggers profound phenotypes and devastating monogenic diseases, loss/mutation of RGS proteins yields more subtle phenotypes, making their contribution to disease harder to study. In this body of work, I define new analytical methods for predicting functionally-relevant variants in human RGS proteins, which participate in complex, polygenic diseases. I then extend these analyses to RGS14, a regulator of long term potentiation and spatial learning. I uncover two human RGS14 variants that disrupt its nuclear-cytoplasmic compartmental balance, and impede its capacity to suppress long term potentiation. To further explore the unique cellular and subcellular localization of human RGS14, we immunolabeled native RGS14 in monkey and human brain, and found broader expression compared to rodents, in brain regions including the basal ganglia (striatum, globus pallidus, and substantia nigra), amygdala, and hippocampus. At the subcellular level, a population of nuclear RGS14 was found, corroborating my findings in neuronal cultures. These studies, combined with my work examining the contribution of human variants tipping the nucleo-cytoplasmic balance, suggest that human carriers may see a loss-of-function of RGS14 in cytosolic compartments such as spines and dendrites, and/or a gain-of-function of RGS14 in the nucleus. The widespread distribution of RGS14 in human brain further suggests that this disequilibrium may have detrimental effects in previously unexplored ways within the basal ganglia and beyond. Overall, I propose RGS14 is a native nucleo-cytoplasmic shuttling protein in the brain, and that human RGS14 has unique localization and sequence diversity uncaptured by previous mouse studies.
Table of Contents
CHAPTER 1: INTRODUCTION: REGULATORS OF G PROTEIN SIGNALING ARE CRITICAL MEDIATORS OF HUMAN PHYSIOLOGY, TRAITS, AND DISEASE................................................................................ 1
1.1. G protein Signaling............................................................................................ 2
1.2 A (Very) Brief History of Regulators of G Protein Signaling....................... 2
1.3 RGS Protein Rare Human Variants in Complex Diseases................................ 3
1.4 RGS Proteins in Physiology and Human Disease.............................................. 8
1.5 Pharmacological Impact of Human Variants in RGS Proteins.................... 24
CHAPTER 2: GENETIC ANALYSIS OF REGULATORS OF G PROTEIN SIGNALING (RGS) PROTEINS 29
2.1 Analysis of Rare Human Variants of RGS Proteins...................................... 30
2.2 Methods............................................................................................................. 33
2.3 R4 Family: RGS4 Rare Variants....................................................................... 35
2.4 R7 Family: RGS9 Rare Variants....................................................................... 38
2.5 R12 Family: RGS10 Rare Variants................................................................... 41
2.6 RZ Family: RGS17 Rare Variants..................................................................... 44
2.7 RGS4 HUMAN VARIANT: PROOF-OF-PRINCIPLE............................................ 48
2.8 DISCUSSION...................................................................................................... 48
CHAPTER 3: RARE HUMAN VARIANTS IN REGULATOR OF G PROTEIN SIGNALING 14 (RGS14) DISRUPT NUCLEO-CYTOPLASMIC EQUILIBRIUM AND INHIBITION OF LONG TERM POTENTIATION IN HIPPOCAMPAL NEURONS 53
3.1 ABSTRACT......................................................................................................... 54
3.2 INTRODUCTION................................................................................................ 55
3.3 METHODS.......................................................................................................... 57
3.4 RESULTS............................................................................................................ 61
DISCUSSION............................................................................................................ 76
CHAPTER 4: REGULATOR OF G PROTEIN SIGNALING 14 (RGS14) IS EXPRESSED PRE- AND POSTSYNAPTICALLY IN NEURONS OF HIPPOCAMPUS, BASAL GANGLIA, AND AMYGDALA OF MONKEY AND HUMAN BRAIN 79
4.1 ABSTRACT......................................................................................................... 80
4.2 INTRODUCTION................................................................................................ 81
4.3 MATERIALS and METHODS............................................................................. 83
4.4 RESULTS............................................................................................................ 89
4.5 DISCUSSION.................................................................................................... 107
4.6 CONCLUSIONS................................................................................................ 115
CHAPTER 5: DISCUSSION: RGS14 IS A MULTIFUNCTIONAL NUCLEO-CYTOPLASMIC SHUTTLING PROTEIN WHOSE LOCALIZATION IS DISRUPTED BY HUMAN GENETIC VARIANTS........................ 117
5.1 INTRODUCTION.............................................................................................. 118
5.2 RGS14: CROSSTALK BETWEEN SEQUENCE DIVERSITY AND LOCALIZATION 121
5.3 WORKING MODEL.......................................................................................... 127
5.4 FUTURE DIRECTIONS..................................................................................... 129
5.5 CONCLUDING REMARKS............................................................................... 130
APPENDIX: REGULATOR OF G PROTEIN SIGNALING 14 (RGS14) FORMS A HETEROMERIC G PROTEIN COMPLEX: INTERACTION BETWEEN RAP2A, H-RAS, AND Gai1.................................................. 132
A.1 INTRODUCTION............................................................................................. 133
A.2 MATERIALS and METHODS.......................................................................... 134
A3. RESULTS......................................................................................................... 136
A.4 DISCUSSION.................................................................................................... 145
REFERENCES 148
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