Activation and regulation of the brain-expressed adhesion G protein-coupled receptors ADGRB1/BAI1 and ADGRB2/BAI2: Implications for human disease Pubblico

Abstract

The adhesion G protein-coupled receptors (GPCRs) are the second-largest family of GPCRs in the human genome. Most adhesion GPCRs are considered to be orphan receptors with few widely-accepted natural ligands. However, the unique architecture and common domain structures of these receptors invites the hypothesis that they may have a common activation mechanism. While the activation and signaling mechanisms of these receptors largely remain enigmatic, even less is understood in terms of how these proteins are regulated.

The first third of this dissertation focuses on progress in understanding the activation mechanisms of the adhesion GPCR BAI1/ADGRB1 (B1). We found that removing almost the entire extracellular amino terminus of the receptor has no negative effect on the signaling activity of the receptor, with the heavily truncated receptor being recognized by β-arrestins and G proteins as an active receptor conformation. Thus, contrary to the tethered cryptic agonist model that has been proposed based on studies of several other aGPCRs, activation of B1 does not appear to require a tethered cryptic agonist.

Next, we applied similar methods to study a human disease-associated mutation in the closely-related receptor BAI2/ADGRB2 (B2). This mutation was found in an adult female patient who has suffered from a degenerative neuromuscular condition since adolescence. We found that this arginine to tryptophan (R1465W) substitution in the intracellular C terminus of B2 significantly increases the receptor's signaling output, but only in the active form of the receptor that has a truncated N-terminus (B2ΔNT). This mutation also increases receptor surface expression, and our results suggest two mechanisms underlying these differences: increased flexibility of G protein coupling and disruption in the receptor's interaction with the endocytic protein endophilin A1.

In the final third of this dissertation, studies are presented on regulation of B1 signaling by C-terminal determinants. We found that deletion of the B1 proline-rich region drastically biases the receptor's signaling activity by eliminating G protein-dependent signaling to SRF-luciferase and greatly potentiating activity to NFAT. Finally, we report on the identification of three mutations in B1 that were discovered in individuals with schizophrenia. One of these mutations is in a similar location as the B2-R1465W, is also an Arg to Trp substitution, and greatly increases receptor activity.

Taken together, these studies provide a deeper understanding of two adhesion GPCRs in terms of their signaling and regulation. Moreover, the findings described here provide a basis to connect the activity of these receptors to human diseases and set the stage for the eventual targeting of these receptors by therapeutics.

Table of Contents

Chapter 1. Introduction
1.1 Cell surface receptors 2
1.2 Key features of GPCRs 7
1.3 Regulation of GPCR signaling activity 8
1.4 Mutations in GPCRs can cause human diseases 15
1.5 Adhesion GPCR family 16
1.5.1 Adhesion GPCR structure 20
1.6 Adhesion GPCR activation mechanisms 25
1.6.1 Adhesion GPCR Signaling Pathways and Ligands 32
1.6.2 Adhesion GPCR Ligands 36
1.7 Adhesion GPCRs in disease 39
1.7.1 Neuropsychiatric disorders 39
1.7.2 Cancer 42
1.7.3 Immune, Cardiac, and Pulmonary 45
1.8 The BAI sub-family of adhesion GPCRs 48
1.9 Research aims 51

Chapter 2. ADGRB1 activation - is there a tethered agonist?
2.1 Introduction 55
2.2 Experimental procedures 58
2.2.1 Cell culture 58
2.2.2 DNA Constructs 58
2.2.3 Western Blot 58
2.2.4 Cell Surface Biotinylation 59
2.2.5 Co-immunoprecipitation 60
2.2.6 Luciferase assays 60
2.3 Results 61
2.3.1 Removal of the NT does not impair receptor expression or signaling 61
2.3.2 B1ΔNT and B1-SL bind to -arrestin2 and are ubiquitinated 66
2.3.3 G13 binds to B1ΔNT and B1-SL and potentiates their signaling activity 71
2.4 Discussion of ADGRB1 activation mechanisms 76

Chapter 3. A disease-associated C-terminal mutation in ADGRB2 potentiates receptor signaling activity
3.1 Introduction 82
3.2 Experimental procedures 85
3.2.1 Cell culture 85
3.2.2 DNA constructs 85
3.2.3 Western Blot 86
3.2.4 Cell Surface Biotinylation 86
3.2.5 Co-immunoprecipitation 87
3.2.6 Luciferase assays 87
3.3 Results 88
3.3.1 ADGRB2 R1465W 88
3.3.2 ADGRB2 R1465W increases signaling activity and surface expression 90
3.3.3 B2ΔNT signals to NFAT luciferase via Gβγ and a calcium channel 93
3.3.4 B2 is linked to Gαz 96
3.3.5 R1465W mutation disrupts B2 interaction with Endophilin A1 102
3.4 Discussion of ADGRB2 R1465W 110

Chapter 4. C-terminal determinants of ADGRB1 activity
4.1 Introduction 117
4.2 Experimental procedures 123
4.2.1 Cell culture 123
4.2.2 DNA constructs 123
4.2.3 Western Blot 124
4.2.4 Cell Surface Biotinylation 124
4.2.5 Co-immunoprecipitation 125
4.2.6 Luciferase assays 125
4.2.7 Mutation discovery 126
4.3 Results 127
4.3.1 IRSp53 increases surface expression and activity of ADGRB1 127
4.3.2 Proline-rich region deletion does not interrupt IRSp53 binding but drastically biases B1 signaling activity 131
4.3.3 ADGRB1 SZ-linked mutations 137
4.4 Discussion of ADGRB1 C terminal determinants of signaling activity 145

Chapter 5. Summary Discussion and Future Directions
5.1 Summary of advances 150
5.2 Limitations 154
5.3 Future Directions 155
5.3.1 Drug targeting opportunities in adhesion GPCRs 158
5.4 Conclusion 162
References 163

List of Figures:
Figure 1. History of receptor theory 6
Figure 2. Regulation of GPCR signaling activity 10
Figure 3. Adhesion GPCR structure 19
Figure 4. Adhesion GPCR sub-families 24
Figure 5. Models of adhesion GPCR activation 29
Table 1. Tethered agonist-activated adhesion GPCRs 31
Table 2. aGPCR signaling pathways 35
Figure 6. The BAI sub-family of adhesion GPCRs 47
Figure 7. B1-SL is expressed on the cell surface 63
Figure 8. ADGRB1 N terminus is dispensable for signaling activity 64
Figure 9. ADGRB1ΔNT and B1-SL bind to -arrestin2 67
Figure 10. ADGRB1ΔNT and B1-SL are heavily ubiquitinated 69
Figure 11. ADGRB1ΔNT and B1-SL bind to G13 72
Figure 12. Gα12 and Gα13 potentiate signaling activity to NFAT luciferase 73
Figure 13. Allosteric antagonist model 75
Figure 14. ADGRB2 R1465W 89
Figure 15. ADGRB2 R1465W increases signaling activity 91
Figure 16. B2 R1465W increases surface expression 92
Figure 17. B2ΔNT signals to NFAT-luciferase via Gβγ and a calcium channel 94
Figure 18. ADGRB2 interacts with Gαz 98
Figure 19. Gαz-specific inhibitor RGS20 amplifies B2ΔNT signaling 100
Figure 20. B2ΔNT-R1465W interacts with Gαi 101
Figure 21. B2ΔNT interacts with β-arrestin2 104
Figure 22. B2ΔNT-WT and B2ΔNT-RW are each heavily ubiquitinated 105
Figure 23. R1465W mutation disrupts interaction with endophilin A1 106
Figure 24. Endophilin A1 inhibits B2ΔNT signaling activity 107
Figure 25. Endophilin A1 interacts with B2 and ADGRB3 in mouse brain 108
Figure 26. ADGRB2 signaling pathway model 109
Table 3. Adhesion GPCR cytoplasmic domain features 122
Figure 27. IRSp53 improves the surface expression of B1ΔNT 128
Figure 28. IRSp53 selectively increases the activity of B1ΔNT to NFAT-luciferase 129
Figure 29. Loss of proline-rich region does not abrogate IRSp53 binding 133
Figure 30. B1ΔNTΔPRR co-immunoprecipitates with endophilin A1 134
Figure 31. PRR loss silences SRF but potentiates NFAT activity 135
Figure 32. B1 schizophrenia (SZ)-associated mutations 138
Figure 33. Surface expression of B1 schizophrenia (SZ)-associated mutants 140
Figure 34. R1473W increases B1ΔΝT signaling activity 141
Figure 35. B1 R1473W retains interactions with endophilin A1 143
Figure 36. Effects of C-terminal mutations on B1 signaling 144
Figure 37. Adhesion GPCR targeting strategies 161

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Health Sciences, Pharmacology
Parola chiave	Endophilin G protein mutation GPCR
Committee Chair / Thesis Advisor	Hall, Randy A, Emory University
Committee Members	Hepler, John R, Emory University Kukar, Thomas, Emory University Bassell, Gary, Emory University Van Meir, Erwin, Emory University

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