Amyloid Precursor Protein Regulation by G Protein-Coupled Receptor 12 Público

Abstract

Alzheimer’s disease (AD) causes irreversible neurodegeneration and does not have a cure. Its pathology is characterized in part by the production of amyloid-β (Aβ) through deleterious processing of amyloid precursor protein (APP). G protein-coupled receptors (GPCRs) make up the largest and most successful family of drug targets and have been shown to modulate APP processing in AD pathology. G protein-coupled receptor 12 (GPR12) has become a receptor of interest because it has recently been implicated in neurological disease, and it shares >60% homology with G protein-coupled receptor 3 (GPR3), which has been shown to interact with APP and increase Aβ production. Given its significant homology to GPR3, GPR12, which has not been studied before in the context of AD, may regulate APP processing and prove to be a potential drug target for AD treatment. In this project, we sought to explore a potential role for GPR12 in APP processing, with the specific aim of elucidating the mechanisms through which GPR12 may be regulating APP expression. GPR12 was co-expressed with APP in HEK-293T cells, and protein levels of APP were examined via Western blot to determine the effect of GPR12 on overall APP expression levels. Co-immunoprecipitation experiments were also conducted to examine protein-protein interaction between APP and wild-type or truncated versions of GPR12. Lastly, luciferase assays were conducted to examine the effects that co-expression of GPR12 and APP have on GPR12 signaling. We found GPR12 to be a novel regulator of APP levels, as co-expression with APP in HEK-293T cells resulted in a dramatic increase in total APP levels. Furthermore, we also identified GPR12 as a novel APP binding partner, and preliminary data from signaling assays revealed that APP can reciprocally affect GPR12 function and signaling. Taken together, these results suggest that GPR12 plays a significant role in AD pathogenesis and could thus serve as a potential drug target for novel AD therapeutics.

Table of Contents

Table of Contents

Abstract …………………………………………………………………………………..……........................................................… 3

Introduction …………………………………………………………………….......................................................……………….. 7

Methods ……………………………………………………………………………......................................................……….....… 10

Results …………………………………………………………………...…......................................................……………..…..... 13

Discussion ………………...………………………………………......................................................……………………..…...… 19

Tables and Figures …………….………………………………………………………......................................................….…... 25

Figure 1: Co-expression of GPR12 with APP-Swe causes a robust increase in full-length APP expression…………………………………………………….................................................................................................. 25

Figure 2: Co-expression of GPR12 with APP leads to APP dimerization……...................................................……... 26

Figure 3: Administration of Compound E successfully enhances expression of CTFs…………………………………….. 27

Figure 4: GPR12 alters the ratio of full-length APP to APP CTF production……...............................................….… 28

Figure 5: GPR12 forms a protein complex with APP……………………...……......................................................…….. 29

Figure 6: Complex formation between GPR12 and APP is not dependent on the N-terminus or the C-terminus

plus the last two transmembrane domains of GPR12….........................................................................................… 30

Figure 7: Truncated versions of GPR12 do not alter APP expression significantly nor do they cause APP dimerization………………………………………….....................................................................................…………...…… 31

Figure 8: Other Gs signaling GPCRs do not alter APP expression to the same extent as GPR12……………………….. 32

Figure 9: Co-expression of GPR12 with APP leads to increased Gs signaling…...................................................….. 33

Figure 10: Models for how APP is increasing GPR12 activity through Gs signaling…............................................... 34

References ………………………………………………………………………………..........................................................…… 35

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Neuroscience
Palavra-chave	G Protein-Coupled Receptor 12 Amyloid Precursor Protein Alzheimer's Disease
Committee Chair / Thesis Advisor	Randy A. Hall, Emory University
Committee Members	Arri Eisen, Emory University Thomas Kukar, Emory University

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