Investigating Fusion Machinery in G Protein-Coupled Receptor Exocytosis
Chen, Hao
2024
Abstract
Vesicle fusion at the plasma membrane plays an essential role in the release of secretory molecules such as neurotransmitters and in the exocytosis of surface proteins such as GPCRs. For neurotransmitter release, vesicle fusion is mediated by a multi-protein machinery including the SNARE complex consisting of vesicle-localized v-SNAREs, called VAMPs, and their cognate t-SNAREs. Surprisingly, in contrast to the well-studied neurotransmitter release, it is largely unclear how fusion machinery is involved in GPCR exocytosis. GPCRs are signal-transducing receptors that sense and respond to extracellular stimuli. To allow for adaptive responses to the everchanging extracellular environment, the trafficking of GPCRs is strictly regulated. We sought to investigate the missing role fusion machinery plays in GPCR exocytosis by hypothesizing that there might be subtype-dependent specificity of v-SNAREs in the exocytosis of different GPCRs. In this dissertation, we focused on three receptors including two prototypical GPCRs, MOR and B2AR, and a non-GPCR trafficking model TfR. Evidence has shown that these receptors undergo distinct exocytic pathways. First, we examined the specificity of VAMP2, a prototypical v-SNARE known to mediate exocytotic vesicle fusion, in GPCR recycling. Using high-speed multi-channel microscopy to visualize VAMP2 and receptors in fusion events simultaneously, we found that VAMP2 was preferentially enriched in vesicles that mediated MOR recycling but not B2AR and TfR. VAMP2 depletion significantly decreased the recycling capacity of MOR on both single cell and population levels and from a cell model where VAMP2 was expressed ectopically to neurons where VAMP2 was expressed endogenously. By contrast, the recycling of B2AR and TfR were not affected by VAMP2 depletion. Interestingly, VAMP2 showed similar subcellular localization on MOR- and B2AR-containing endosomes, suggesting that VAMP2 and MOR were co-packaged into vesicles from the same endosomes that also contained other receptors. These results indicated that VAMP2 was cargo-selective and GPCRs might utilize distinct SNARE assemblies for vesicle fusion. Next, we sought to address whether B2AR depended on a v-SNARE other than VAMP2 for recycling. We examined B2AR recycling under the depletion of VAMP4 or VAMP7 and found neither of them was required. VAMP1 and VAMP3 were also excluded due to their lack of baseline expression. We concluded that there might not be an exclusive v-SNARE involved in B2AR recycling. Finally, we investigated the underlying mechanism leading to VAMP2’s cargo-selectivity by hypothesizing that VAMP2 might possess an unknown sorting sequence through which it was co-sorted to endosomal subdomains alongside MOR. Through a series of microscopy studies, we found VAMP2 showed a sub-endosomal localization distinct from VAMP7. To follow up on our results of VAMP2’s MOR-selectivity, we generated VAMP2 truncation mutants and studied their involvement in MOR recycling. Interestingly, both the N-terminus (V2N) and SNARE motif (V2S) of VAMP2 were required for the MOR-selectivity. By studying chimeric VAMP7 transplanted with VAMP2’s protein sequences, we found that although V2N alone was sufficient to transform VAMP7’s sorting, full transformation only occurred when V2S was also transplanted. Moreover, V2S was required to maintain VAMP2’s stable surface localization. These data indicated that V2N and V2S played critical but distinct roles in the sorting and trafficking of VAMP2. Together, our results revealed a previously unclear mechanism in GPCR exocytosis and demonstrated another layer of regulation on GPCR trafficking. Moreover, our results provided evidence to a novel mechanism of v-SNARE endosomal sorting and contributed to an improved understanding of cellular trafficking.Deep Blue DOI
Subjects
G protein-coupled receptors (GPCRs) Exocytosis Fusion machinery SNARE proteins Vesicular trafficking Microscopy
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