Phosphatidylserine Exposure Modulates GPCR BAI1 (ADGRB1) Signaling Activity 公开

Abstract

The adhesion G protein-coupled receptor family consists of several dozen receptors containing massive N-terminal fragments (NTFs), which confer these receptors with adhesive properties. Recent advances in the field have revealed that some family members can be modulated by small molecules, mechanosensory forces and adhesive ligands, suggesting that these receptors function as massive integrators of multimodal signaling. 

 

The body of work described here provides new insights into the signaling properties of the adhesion GPCR BAI1 (also known as ADGRB1 or “B1”). This receptor has previously been shown to bind phosphatidylserine (PS) via its NTF, but it remains unknown whether this interaction alters the signaling activity of the receptor. B1 in most highly expressed in the brain and can couple to G proteins and other pathways to regulate postsynaptic function and dendritic spine morphology. We investigated G protein-dependent signaling by B1 in the absence and presence of the PS-flippase ATP11A, which can modulate the amount of PS available for B1 to bind in the outer leaflet of the plasma membrane. We mainly used the HEK293T cell system for these studies, as we found that these cells exhibit a quantifiable baseline level of PS exposure that can be modulated by ATP11A overexpression. We observed that ATP11A expression dramatically reduced B1 G protein-dependent signaling for the wild-type receptor but not a truncated mutant lacking the large extracellular NTF, suggesting that the NTF of B1 is required for PS sensing. The flippase activity of ATP11A was found to be essential for regulation of B1 signaling, and co-immunoprecipitation experiments revealed that ATP11A not only modulates B1 signaling but also forms complexes with B1. To characterize regulation of B1 signaling by PS exposure using an independent method, we studied B1 signaling in cells with lower PS externalization due to deletion of the endogenous PS scramblase ANO6 and found that this manipulation also resulted in lowered B1 signaling activity. These findings demonstrate that B1 signaling is modulated by PS exposure and therefore implicate B1 as a PS sensor at synapses and in other cellular contexts. 

 

The studies described here provide a deeper understanding of the adhesion GPCR B1 and also contribute to a deeper understanding of NTF-mediated regulation of signaling by other AGPCRs. Additionally, this work elucidates how changes in PS exposure can be detected in the brain, which has implications for synaptic pruning, synaptic plasticity, and other brain processes known to be regulated by externalization of PS.

Table of Contents

Chapter 1: Introduction

1.1        CELL-CELL COMMUNICATION

1.1.1 Cell surface receptors

1.1.2 Synaptic communication

1.1.3 Overview of GPCRs

1.2 GPCR MULTIMERIZATION

1.2.1 GPCR Allosteric Modulation

1.3 ADHESION GPCR FAMILY

1.3.1 AGPCR Nomenclature

TABLE 1: Annotated names and chromosomal locations of AGPCRs

1.3.2 AGPCR Structure

Figure 1.1 AGPCRs exhibit great structural diversity.

1.3.2.1 Autoproteolysis of AGPCRs

1.3.3 Signaling of AGPCRs

1.3.3.1 Canonical G protein-dependent signaling

Figure 1.2.  AGPCRs engage in diverse signaling mechanisms.

1.3.3.2 Non-canonical G protein-independent signaling of AGPCRs

TABLE 1.2: AGPCR G protein-dependent and alternate signaling pathways

1.3.4 Current hypotheses behind AGPCR G protein-dependent activation

1.3.4.1 Tethered agonism

1.3.4.2 Beyond tethered agonism

Figure 1.3.  There are multiple mechanisms by which AGPCRs can be activated.

1.3.5 AGPCRs as multimodal signal-transducing platforms

Figure 1.4.  AGPCRs can engage with a diverse array of ligands to exert their physiological actions.

TABLE 1.3: AGPCR-ligand binding and physiological significance

1.3.5.1 Mechanosensory signaling

1.3.5.2 Small molecule ligands

Figure 1.5.  AGPCRs can integrate heterogeneous signals.

1.3.6 BAI family of AGPCRs

1.3.6.1 BAI family physiology

1.3.6.1.1. Immune System

Figure 1.6. B1-mediated engulfment of apoptotic cells.

1.3.6.1.2 Nervous System

Figure 1.7.  B1 can couple to multiple signaling pathways with differential effects on physiology.

1.3.6.1.3 Muscular System

1.3.6.2 BAI family pathophysiology

1.4 Fluid mosaic model and lipid asymmetry

1.4.1 Significance of phosphatidylserine

1.4.3 PS exposure modulation

1.4.3.1 Flippases

1.4.3.2 Scramblases

Figure 1.8.  Flippases and Scramblases regulate phospholipid asymmetry in the plasma membrane.

1.5 Research aims

Chapter 2: Modulation of Phosphatidylserine Exposure Regulates B1 Signaling Activity

2.1 Phosphatidylserine Exposure Modulates Adhesion GPCR BAI1 (ADGRB1) Signaling Activity

2.1.1 Introduction

2.1.2 Results

2.1.2.1 HEK293T cells exhibit a baseline level of exposed PS that can be modulated by the PS flippase ATP11A

Figure 2.1. Evaluation of phosphatidylserine exposure in HEK293T cells at baseline and when overexpressing ATP11A.

2.1.2.2 Coexpression of ATp11A with B1 reduces the constitutive signaling activity of B1

Figure 2.2. ATP11A coexpression reduces B1 signaling activity.

2.1.2.3 The flippase activity of ATP11A is required for modulation of B1 signaling

2.1.2.4 Increased cell density does not promote B1 signaling

Figure 2.3. Flippase-null mutant ATP11A (E186Q) does not alter B1 signaling activity.

2.1.2.5 B1 multimerizes via its transmembrane domains in a PS-independent manner

2.1.2.6 B1 interacts via its NTF region with ATP11A

Figure 2.4. B1 forms PS-independent multimers and also interacts with ATP11A.

2.1.2.7 B1 signaling is reduced in cells lacking the scramblase ANO6

Figure 2.5. B1 signaling activity is reduced in cells lacking ANO6.

2.1.3 Discussion

2.1.4 Experimental Procedures

2.1.3.1 Constructs

2.1.3.2 Cell culture

2.1.3.3 Luciferase reporter assay

2.1.3.4 Co-culture experiments

2.1.3.5 Western blot

2.1.3.6 Co-immunoprecipitation

2.1.3.7 Cell surface biotinylation

2.1.3.8 Flow cytometry

2.1.3.9 Quantification and data analysis

Chapter 3: Discussion and Future Directions

3.1 Summary of Advances

Figure 3.1: Proposed mechanism for B1 interaction with PS and subsequent impact of ATP11A on B1 signaling.

3.2 Is PS the Only Ligand for B1?

Figure 3.2: B1 ligands that bind the receptor NTF are varied and trigger diverse processes in different cellular contexts.

3.3 Limitations

3.3 Future Directions

3.4 PS regulation of synaptic pruning

Figure 3.3: B1 presence in spines is protective and without it, PS is dysregulated and makes the spine susceptible to microglial engulfment.

3.5 Conclusion

References

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
关键词	GPCR Phosphatidylserine Synaptogenesis BAI1
Committee Chair / Thesis Advisor	Ellen Hess, Emory University Randy Hall, Emory University
Committee Members	John Hepler, Emory University James Zheng, Emory University Victor Faundez, Emory University

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