GPR37 & GPR37L1: LIG and Identification, Cell Protective Signaling & Dopaminergic Cross-Talk Open Access

Meyer, Rebecca C. (2014)

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G protein-coupled receptors (GPCRs) are essential to cellular communication in the central nervous system and constitute one of the largest classes of drug targets in the body. The ligands for many GPCRs are known, but there remains a subset of orphan GPCRs for which ligands have yet to be identified. These orphan receptors have the potential to be novel therapeutic targets; however, identification of endogenous ligands for orphan receptors is a crucial step toward therapeutically targeting these receptors. Here, we present evidence that the orphan GPCRs GPR37 and GPR37L1 can be activated by prosaposin, a neuroprotective and glioprotective factor, and by the active fragment of prosaposin, prosaptide. We found that prosaptide and prosaposin bind to GPR37 and GPR37L1 to promote receptor signaling and internalization. Prosaptide and prosaposin stimulation of cells transfected with GPR37 or GPR37L1 induced increases in ERK phosphorylation and reductions in cAMP levels in a pertussis toxin-sensitive manner, indicating that the receptors are Gi-coupled. Work in cultured astrocytes in which GPR37 and/or GPR37L1 were depleted via siRNA knockdown indicated that prosaptide induces ERK phosphorylation through stimulation of endogenous GPR37. Additionally, we identified that GPR37-mediated ERK phosphorylation occurs through transactivation of the EGF receptor. Furthermore, we found that both GPR37 and GPR37L1 contribute to the ability of prosaptide and prosaposin to protect cortical astrocytes against oxidative cell death. In additional studies, we identified a physical interaction between GPR37L1 and the dopamine D1 receptor. Furthermore, co-expression of these receptors altered D1-mediated dopamine stimulation of cAMP and pERK, indicating the potential for GPR37L1 to regulate dopaminergic signaling. In conclusion, the work presented here describes both ligand-dependent and potentially ligand-independent functions for GPR37 and GPR37L1 as protective receptors with the ability to influence dopaminergic function. Their ligand, prosaposin, has previously been shown to be neuroprotective in animal models of Parkinson's disease and focal cerebral ischemia, and has also been shown to promote nerve remyelination after injury. Thus, establishing GPR37 and GPR37L1 as receptors for prosaptide and prosaposin identifies novel therapeutic targets for the treatment of neurodegeneration and myelination disorders.

Table of Contents

CHAPTER 1: Introduction to G Protein-coupled Receptor Function and Signaling. 1

Section 1.1: G Protein-coupled Receptors. 2

1.1.1 GPCR Signaling. 2

1.1.2 GPCRs as Pharmaceutical Targets. 3

1.1.3 GPCR Heterodimerication as a Mechanism for Signaling Regulation. 10

1.1.4 Orphan G Protein-coupled Receptors. 11

Section 1.2: GPR37 and GPR37L1. 13

1.2.1 Expression and Receptor Homology. 13

1.2.2 Physiological Function and Role in Neuropathology. 15

Section 1.3: Prosaposin and Prosaptide. 17

1.3.1 Prosaposin as a Lysosomal Protein. 17

1.3.2 Prosaposin as a Secreted Factor. 20

1.3.3 Prosapsoin Exocytosis Following Injury/Stress. 23

1.3.4 Mechanisms Controlling Prosaposin Release. 23

Section 1.4: Effects of Secreted Prosaposin in the Nervous System. 24

1.4.1 Prosaposin Rescues Ischemic Damage. 24

1.4.2 Prosaposin Rescues Dopaminergic Neurons. 25

1.4.3 Prosaposin Facilitates Nerve Regeneration and Alleviates Sensory Neuropathy. 26

1.4.4 Prosaposin Protects Myelinating Glial Cells. 26

1.4.5 Prosaposin Influences Cerebellar Development and Survival. 27

1.4.6 Prosaposin Protects Diverse Cell Types from Cellular Insults. 28

Section 1.5: Receptors Controlling Prosaposin Uptake. 28

1.5.1 Importance of Prosaposin Uptake. 28

1.5.2 Prosaposin Uptake Mediated by LRP1. 29

Section 1.6: Receptors Mediating Prosaposin Signaling via G Proteins. 30

1.6.1 Prosaposin Stimulates ERK and Akt Phosphorylation. 30

1.6.2 Prosaposin Can Stimulate G Protein-Mediated Signaling. 33

Section 1.7: Dissertation Goals. 33

CHAPTER 2: Identification of Prosaptide and Prosaposin as Ligands for GPR37 and GPR37L1 in Transfected Cells. 37

Section 2.1: Rationale. 38

Section 2.2: Experimental Methods. 39

2.2.1 Materials. 39

2.2.2 Cell Culture. 40

2.2.3 Prosaposin Production. 41

2.2.4 Luminometer Assay. 42

2.2.5 Western Blotting. 42

2.2.6 Biotinylated Prosaptide Pulldown. 43

2.2.7 35S-GTPγS Binding. 44

2.2.8 cAMP Signaling. 45

2.2.9 pERK Signaling. 45

Section 2.3: Results. 46

2.3.1 Prosaptide, but not other Peptides, Induces the Internalization of GPR37 and GPR37L1 46 2.3.2 Prosaptide does not induce Internalzation of Other Related Receptors. 47

2.3.3 Prosaptide Binds to GPR37 and GPR37L1, but Not Other GPCRs. 50

2.3.4 Prosaptide Stimulates 35S-GTPγS Binding to GPR37 and GPR37L1 Co-Transfected with Gαi. 50

2.3.5 GPR37 and GPR37L1 Mediate the Ability of Prosaptide to Inhibit cAMP Production. 53

2.3.6 GPR37 and GPR37L1 Mediate the Ability of Prosaptide to Induce ERK Phosphorylation. 55

2.3.7 Prosaptide Induction of pERK Through GPR37 and GPR3L1 is Pertussis Toxin-Sensitive. 60

2.3.8 Prosaposin is able to Stimulate Internalization and ERK Phosphorylation through GPR37 and GPR37L1. 60

Section 2.4: Summary and Discussion. 66

CHAPTER 3: Prosaptide and Prosaposin Induce Pro-Survival Signaling in Cortical Astrocytes via Stimulation of GPR37 and GPR37L1. 68

Section 3.1: Rationale. 69

Section 3.2: Experimental Methods. 70

3.2.1 Materials. 70

3.2.2 Cell Culture. 70

3.2.3 Prosapsoin Production. 71

3.2.4 siRNA Knockdown. 72

3.2.5 pERK Assay. 72

3.2.6 Cytotoxicity. 73

3.2.7 Western Blotting. 73

Section 3.3: Results. 74

3.3.1 Prosaptide and Prosaposin Induced ERK Phosphorylation Through GPR37, but not GPR37L1, in Cortical Astrocytes. 74

3.3.2 GPR37 and GPR37L1 Mediate the Ability of Prosaptide and Prosaposin to Protect Against Cell Death Induced by Oxidative Cell Stress In Cortical Astrocytes. 80

3.3.3 GPR37 and GPR37L1 Mediate the Pro-Survival Effects of Prosaptide and Prosaposin Effects Partially Through Preventing the Cleavage of Procaspase-3. 83

3.3.4 Knockdown of GPR37 and GPR3L1 Does Not Alter Fundamental Cortical Astrocyte Signaling or Basal Cell Death from Hydrogen Peroxide. 85

3.3.5 GPR37 Mediates Prosaptide Signaling to pERK through Transactivation of the EGF Receptor 87 Section 3.4: Summary and Discussion. 94

CHAPTER 4: Functional Heterodimerization of GPR37L1 and the Dopamine D1 Receptor 97 Section 4.1: Rationale.98

Section 4.2: Experimental Methods. 99

4.2.1 Materials. 99

4.2.2 Cell Culture. 100

4.2.3 Luminometer Assay. 100

4.2.4 Co-Immunoprecipitation. 101

4.2.5 Western Blotting. 102

4.2.6 cAMP Assay. 103

4.2.7 pERK Assay. 103

Section 4.3: Results. 104

4.3.1 The Dopamine D1 Receptor Preferentially Co-Immunoprecipitates with GPR37L1 over GPR37 104 4.3.2 Ligand-Stimulated Cross-Internalization of GPR37L1 and D1. 107

4.3.3 Co-Expression with GPR37L1, but Not GPR37, alters the ability of Dopamine to Stimulate cAMP Production through the D1 Receptor. 110

4.3.4 Co-Expression with GPR37L1 Enhances ERK Phosphorylation Mediated by Dopamine. 111

Section 4.4: Summary and Discussion. 117

CHAPTER 5: Further Discussion and Future Directions. 121

Section 5.1: Summation of Dissertation Work. 122

Section 5.2: Theoretical Model of Prosaposin Function. 124

Section 5.3: Relevance of Cross-Talk between GPR37, GPR37L1, and the Dopamine Receptors. 126

Section 5.4: GPR37 and GPR37L1 as Pharmaceutical Targets. 128

5.4.1 Treatment of Parkinson's disease. 128

5.4.2 GPR37 and GPR37L1 as Pharmaceutical Targets to Treat Parkinson's disease. 130

5.4.3 GPR37 and GPR37L1 as Pharmaceutical Targets for the Treatment of Ischemia. 132

5.4.4 GPR37 and GPR37L1 as Pharmaceutical Targets to Repair Peripheral Nerve Injury. 133

5.4.5 GPR37 and GPR37L1 as Pharmaceutical Targets to Treat Multiple Sclerosis. 134

Section 5.5: Future Directions. 135

5.5.1 Future Directions of the GPR37L1 and D1 Heterodimerization. 135

5.5.2 Future Directions to Study GPR37 and GPR37L1 as Prosaposin Receptors. 136

Section 5.6: Concluding Thoughts. 139

APPENDIX I: Generation of GPR37 and GPR37L1 Double Het Mice. 179

Section I.1: Rationale. 180

Section I.2: Materials and Methods. 180

Section I.3: Results. 182

Section I.4: Conclusions and Discussion. 184

APPENDIX II: Brain Expression of GPR37L1. 185

Section II.1: Rationale. 186

Section II.2: Materials and Methods. 186

Section II.3: Results. 187

Section II.4: Conclusions and Discussion. 190

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