Trafficking and Signaling of Parkin-Associated Endothelin-Like Receptor GPR37 Público

Dunham, Jill Harley (2009)

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Trafficking and Signaling of Parkin-Associated Endothelin Like Receptor

Jill Harley Dunham

Dopaminergic neuronal cell death is a hallmark of Parkinson's disease (PD),
believed at least in part to be due to protein aggregation. This cell death leads to a major
disruption of the dopaminergic system, which is involved in many different aspects of
behavior, such as movement, cognition, motivation, and pleasure. GPR37, also known as
parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled
receptor (GPCR) that exhibits poor plasma membrane expression when expressed in
most cell types. Due to the association of GPR37 with the PD-associated gene parkin,
GPR37 is considered as a potential target for novel PD therapies. Thus, we sought to find
ways to enhance GPR37 trafficking to the cell surface in order to facilitate studies of
GPR37 functional activity in heterologous cells. In truncation studies, we found that
removing the receptor's N-terminus (NT) dramatically enhanced the receptor's plasma
membrane insertion. Further studies on sequential NT truncations revealed that
removal of the first 210 amino acids increased surface expression nearly as much as
removal of the entire NT. In studies examining the effects of co-expression of GPR37
with a variety of other GPCRs, we observed significant increases in GPR37 surface
expression when the receptor was co-expressed with the adenosine receptor A2AR or the
dopamine receptor D2R. Co-immunoprecipitation experiments revealed that full-length
GPR37 and, to a greater extent, the truncated GPR37 were capable of robustly
associating with D2R, resulting in modestly-altered D2R affinity for both agonists and
antagonists. In studies examining potential interactions of GPR37 with PDZ scaffolds,
we observed a specific interaction between GPR37 and syntenin-1, which resulted in a
dramatic increase in GPR37 surface expression in HEK-293 cells. These findings reveal
three independent approaches - N-terminal truncation, co-expression with other
receptors and co-expression with syntenin-1 - by which GPR37 surface trafficking in
heterologous cells can be greatly enhanced to facilitate functional studies and drug
discovery efforts focused on this orphan receptor.



Jill Harley Dunham
B.S., University of Georgia, 2003

Advisor: Randy A. Hall, Ph.D.

A dissertation submitted to the Faculty of the Graduate School of Emory University in
partial fulfillment of the requirements for the degree of Doctor of Philosophy

Molecular and Systems Pharmacology
Graduate Division of Biological and Biomedical Sciences

Table of Contents

Table of Contents

CHAPTER I: Introduction 1
1.1 G-protein coupled receptors (GPCRs) 2
1.2 Clinical relevance of GPCRs 4
1.3 Signaling through GPCRs 6
1.4 Orphan GPCRs 8
1.5 Trafficking of GPCRs 9
1.5.1 Domains enhancing forward trafficking 11
1.5.2 Domains preventing forward trafficking 15
1.5.2 Receptor-receptor interactions that assist in proper trafficking 16
1.5.3 Associating proteins assist in proper trafficking 19
1.5.4 GPCR trafficking defects in human disease 21 Vasopressin and Nephrogenic Diabetes Insipidus 22 Rhodopsin and Retinitis Pigmentosa 23 Gonadotropin-Releasing Hormone Receptor and Hypogonadic Hypogonadism 23
1.5.5 Trafficking of orphan GPCRs 24
1.6 Parkin-Associated Endothelin-Like Receptor (PAEL-R)/GPR37 25
1.6.1 Endothelin-Like 26
1.6.2 Parkinson's disease and the Parkin-Associated Receptor GPR37 29
1.7 Overall Hypothesis 32
CHAPTER II: Structural Determinants Controlling Surface Trafficking of GPR37 34
2.1 Introduction 35
2.2 Experimental Procedures 36
2.3 Results 41
2.3.1 GPR37L1 exhibits robust cell surface expression but GPR37 does not. 41
2.3.2 N-terminal truncation of GPR37 increases plasma membrane expression. 44
2.3.3 Structural determinants on the GPR37 N-terminus control receptor surface expression. 44
2.3.4 Plasma membrane expression of GPR37 does not seem to be influenced by individual N-terminal amino acids. 47
2.3.5 Head Activator does not detectably activate wt GPR37, ΔNT GPR37, Δ1-210GPR37, or GPR37L1. 47
2.4 Discussion 55
CHAPTER III: Regulation of GPR37 Surface Expression by Protein-Protein Interactions 58
3.1 Introduction 59
3.2 Experimental Procedures 62
3.3 Results 68
3.3.1 Antagonists of ETBR do not influence the cellular trafficking of GPR37. 68
3.3.2 Dopamine receptor D2R interacts with GPR37. 68
3.3.3 Co-expression with Δ1-210 GPR37 alters D2R ligand-binding properties. 76
3.3.4 Co-expression of GPR37 with syntenin-1 enhances GPR37 surface expression. 80
3.4 Discussion 83
Chapter IV: Conclusion 87

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