Adhesion G Protein-coupled Receptor G1 (GPR56) Regulation by Sorting Nexin 27 Open Access

Iancu, Ariella Magen (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/p5547s03w?locale=en
Published

Abstract

Adhesion G Protein-coupled Receptor G1 (G1), previously known as GPR56, plays a role in a wide variety of biological functions including cancer, immunology, and development. Mutations in G1 cause the brain developmental disorder Bilateral Fronto-Parietal Polymicrogyria (BFPP), and mutant forms of G1 associated with BFPP exhibit reduced cell surface expression and signaling. Sorting Nexin (SNX) family members typically modulate endosomal trafficking to the plasma membrane, and SNX27 has been recently identified as a potential modulator of G1 in a proteomic study. I showed that SNX27 interacts with both full-length G1 and N-terminally truncated G1 in HEK-293 cells. I also found that SNX27 increases total and surface expression of G1 and N-terminally truncated G1. Preliminary results indicate that SNX27 increases signaling of G1. Based on these results, SNX27 may be an important modulator of G1 expression and signaling in vivo.


Table of Contents

I. Introduction.................................................................................................................................... 1

A. G Protein-coupled Receptors.......................................................................................................... 1

B. Adhesion GPCRs........................................................................................................................... 3

C. Adhesion G Protein-coupled Receptor ADGR-G1 (G1)........................................................................... 5

D. Aims of This Thesis........................................................................................................................ 12

II. Methods.......................................................................................................................................... 13

A. Cell Culture................................................................................................................................... 13

B. Plasmids........................................................................................................................................ 13

C. Antibodies...................................................................................................................................... 13

D. Western Blotting............................................................................................................................. 14

E. Coimmunoprecipitation...................................................................................................................... 14

F. Cell-Surface Biotinylation................................................................................................................... 14

G. Gene Reporter Assay: Dual-Glo Luciferase Assay.................................................................................... 15

H. Statistical Analysis............................................................................................................................. 16

III. Results............................................................................................................................................ 16

A. SNX27 Interacts with G1.................................................................................................................... 16

B. Total and Surface Expression of G1 Increase with SNX27........................................................................ 17

C. SNX27 Increases G1 Signaling............................................................................................................ 20

IV. Discussion....................................................................................................................................... 21

V. Works Cited..................................................................................................................................... 24

Figures

FIGURE 1 SCHEMATIC DIAGRAM OF G1 SIGNALING PATHWAYS..................................................................................................................................... 6

FIGURE 2 ENDOSOMAL SURFACE PROTEIN TRAFFICKING...............................................................................................................................................10

FIGURE 3 PROPOSED SCHEMATIC OF SNX27 INTERACTION WITH G1 LOCALIZED IN THE EARLY ENDOSOME........................................................................... 12

FIGURE 4 SCHEMATIC OF G1 AND dNT VECTORS ........................................................................................................................................................ 16

FIGURE 5 G1 AND dNT INTERACT WITH SNX27............................................................................................................................................................ 17

FIGURE 6 SNX27 INCREASES BOTH TOTAL AND SURFACE EXPRESSION OF G1 AND dNT...................................................................................................... 19

FIGURE 7 QUANTIFICATION OF INCREASE IN G1 TOTAL AND SURFACE EXPRESSION WITH SNX27........................................................................................ 20

FIGURE 8 G1 SIGNALING WITH AND WITHOUT SNX27................................................................................................................................................... 21

About this Honors Thesis

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Degree
Submission
Language
  • English
Research field
Keyword
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files