THESIS
2023
1 online resource (xvi, 103 pages) : illustrations (some color)
Abstract
The enzyme-coupled receptors and G-protein-coupled signaling receptors (GPCRs) are
two major cell surface located receptors that initiate signal transduction. Although
fundamentally important, how these signal receptors after synthesized from ribosomes are
delivered to the cell surface to perform their cellular functions remain largely unclear. My thesis
analyzed molecular mechanisms regulating surface delivery of an enzyme-coupled receptor,
the epidermal growth factor receptor (EGFR), and a GPCR regulating planar cell polarity,
Frizzled6.
EGFR plays an important role in promoting cell differentiation, growth, proliferation
and migration. Newly synthesized EGFR needs to be delivered from the endoplasmic reticulum
(ER) to the cell surface to perform its function. We found that ER export...[
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The enzyme-coupled receptors and G-protein-coupled signaling receptors (GPCRs) are
two major cell surface located receptors that initiate signal transduction. Although
fundamentally important, how these signal receptors after synthesized from ribosomes are
delivered to the cell surface to perform their cellular functions remain largely unclear. My thesis
analyzed molecular mechanisms regulating surface delivery of an enzyme-coupled receptor,
the epidermal growth factor receptor (EGFR), and a GPCR regulating planar cell polarity,
Frizzled6.
EGFR plays an important role in promoting cell differentiation, growth, proliferation
and migration. Newly synthesized EGFR needs to be delivered from the endoplasmic reticulum
(ER) to the cell surface to perform its function. We found that ER export of EGFR depends on
a conserved poly-arginine (polyR) motif on the juxtamembarne region of EGFR cytosolic
domain. Mechanistic analysis indicates that this motif directly interacts with the D198 residue
on SARlA. This interaction regulates the packaging of EGFR into COPII vesicles to be
delivered to the Golgi. Depleting this polyR motif causes defects in EGF-induced activation of
EGFR. Interestingly, we demonstrated that ER export of the oncogenic mutant form of EGFR,
EGFR(L858R), is independent of the D198 residue of SARIA and EGFR polyR motif. Both
wild type EGFR and EGFR(L858R) require ER export for their activations. Our study uncovers
a novel mechanism regulating ER export of EGFR. In addition, our results suggest that ER
export of EGFR(L858R) and wild type EGFR are mediated by different molecular machineries.
Frizzled-6 is an unconventional GPCR regulating planar cell polarity (PCP), which
plays critical role in various developmental processes. Newly synthesized Fzd6 is targeted to
the distal side of plasma membrane to perform its physiological function. Here, we reconstituted
the packaging of Fzd6 into transport vesicles at the trans Golgi network (TGN) and successfully
isolated Fzd6-enriched vesicles. The size of Fzd6-enriched vesicles is significantly larger than
the size of vesicles enriched with another cargo protein, epidermal growth factor receptor
(EGFR). Quantitative mass spechrometry analyses revealed several transmembrane proteins,
including a SNARE protein VTI1B, that are specifically co-emiched with Fzd6 but not EGFR
in TGN-derived vesicles. Mechanistic analyses revealed that VTI1B enhances the interaction
between Fzd6 and its cargo adaptor, epsinR, and promotes the release of Fzd6 into transport
vesicles at the TGN. Functional analyses revealed that VTI1B regulates the TGN-to-cellsurface
delivery of Fzd6. These analyses indicate that VTI1B is an important factor regulating
TGN sorting of Fzd6, suggesting cargo sorting is coupled to vesicle targeting. Another protein
identified in the Fzd6-enriched vesicles, SCAMP2, mediates Fzd6 transpo11 to lysosome for
degradation. In addition, our study established a robust approach to uncover protein profiling
of vesicles enriched with a specific signaling receptor, providing a powerful tool to identify
novel cellular factors regulating vesicular trafficking.
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