Signaling, Regulation, and Synaptic Role of Adhesion G protein-coupled Receptor Brain-specific Angiogenesis Inhibitor-1 Pubblico
Stephenson, Jason (2014)
Abstract
G protein-coupled receptors (GPCRs) are one of the largest gene families in the human genome and major targets for therapeutics. GPCRs recognize a diverse array of extracellular stimuli and transduce intracellular signaling cascades via heterotrimeric G proteins and other intermediates, resulting in modifications to cellular physiology. Brain-specific angiogenesis inhibitor-1 (BAI1) is a member of the adhesion GPCR family that has been studied primarily for its anti-angiogenic and anti-tumorigenic properties, but it has been unknown how BAI1 exerts its effects on cellular physiology or whether this receptor can even couple to G proteins. We found that over-expression of BAI1 in HEK293T cells results in activation of the Rho pathway via a Ga12/13-dependent mechanism, with truncation of the BAI1 N-terminus (NT) resulting in a dramatic enhancement in receptor signaling. This constitutive activity of the truncated BAI1 mutant also resulted in enhanced downstream phosphorylation of extracellular regulated kinase (ERK) as well as increased receptor association with b-arrestin2 and increased ubiquitination of the receptor. To gain insights into the regulation of BAI1 signaling, we screened the C-terminus (CT) of BAI1 against a proteomic array of PDZ domains to identify novel interacting partners. These screens revealed that the BAI1-CT interacts with a variety of PDZ domains from synaptic proteins, including MAGI-3. Removal of the BAI1 PDZ-binding motif resulted in attenuation of the receptor's signaling to Rho, but had no effect on ERK activation. Conversely, co-expression with MAGI-3 was found to potentiate signaling to ERK by constitutively-active BAI1 in a manner that was dependent on the receptor's PDZ-binding motif. Biochemical fractionation studies revealed that BAI1 is highly enriched in post-synaptic density (PSD) fractions, a finding consistent with our observations that BAI1 can interact with PDZ proteins known to be concentrated in the PSD. These findings demonstrate that BAI1 is a synaptic receptor that can activate both the Rho and ERK pathways, with the receptor's NT and CT regions playing key roles in the regulation of BAI1 signaling activity.
Table of Contents
CHAPTER I: Introduction...1
1.1 G protein-coupled receptors...2
1.2 G protein-coupled receptor signaling...4
1.3 Clinical relevance of G protein-coupled receptors...12
1.4 Adhesion G protein-coupled receptors...17
1.5 Adhesion G protein-coupled receptor structure...21
1.6 Adhesion G protein-coupled receptor signaling...23
1.7 Physiological roles of adhesion G protein-coupled receptors....25
1.8 Brain-specific angiogenesis inhibitor subfamily...28
1.9 Autoproteolysis of brain-specific angiogenesis inhibitor subfamily...31
1.10 Physiological roles of brain-specific angiogenesis inhibitor subfamily...33
1.11 Aim of dissertation research...35
CHAPTER II: BAI1 activation of Rho via G proteins and regulation by the N-terminus...38
2.1 Introduction...39
2.2 Experimental procedures...41
2.3 Results...44
2.3.1 BAI1 undergoes autoproteolysis in native brain tissue but not in HEK293T cells...44
2.3.2 The N-terminus interacts with the 7-transmembrane region of BAI1 when co-expressed in HEK293T cells...47
2.3.3 Removal of the BAI1 N-terminus enhances stimulation of Rho via Gα12/13 and cyr61 expression...50
2.4 Discussion...56
CHAPTER III: BAI1 C-terminal interactions and regulation of receptor signaling...60
3.1 Introduction...61
3.2 Experimental procedure...64
3.3 Results...69
3.3.1 BAI1 interacts with a variety of PDZ domains from synaptic proteins...69
3.3.2 Removal of BAI1 PDZ-binding motif attenuates receptor signaling to Rho...72
3.3.3 BAI1 signaling to ERK is potentiated by MAGI-3...75
3.3.4 BAI1 activation of ERK is not mediated by Gα12/13, Gβγ, or transactivation of EGFR...78
3.3.5 Removal of the BAI1 N-terminus enhances association with β-arrestin2 and receptor ubiquitination...82
3.3.6 All BAI1 constructs are expressed equally at the plasma membrane...85
3.4 Discussion...88
CHAPTER IV: BAI1 is a component of the postsynaptic density...92
4.1 Introduction...93
4.2 Experimental Procedure...96
4.3 Results...99
4.3.1 BAI1 is enriched in the postsynaptic density...99
4.3.2 BAI1 co-immunoprecipitates with the postsynaptic density marker PSD-95...102
4.3.3 Genetic knockdown of BAI1 alters expression levels of postsynaptic density associated proteins...105
4.3.4 BAI1 interacts with a domain of the synaptic protein C1ql3...108
4.4 Discussion...111
CHAPTER V: Further discussion and future directions...116
5.1 Signaling and mechanism of activation for BAI1...117
5.2 Possible roles for BAI1 in the postsynaptic density...125
5.3 C1ql3 as a possible endogenous ligand for BAI1...130
5.4 Potential drug development strategies for BAI1...131
5.5 Future directions: further elucidation of the role of BAI1 at the postsynaptic density...132
5.6 Future directions: BAI1 as a drug target for diseases associated with aberrant synapse formation and cancer...134
5.7 Concluding remarks...137
References...140
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