Lateral diffusion contributes to FRET from lanthanide-tagged membrane proteins

Lan, Tien Hung; Wu, Guangyu; Lambert, Nevin A.

doi:10.1016/j.bbrc.2015.06.127

Cited by 11 publications

(9 citation statements)

References 15 publications

(27 reference statements)

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“…To gain more kinetic information on agonist-induced GLP-1R clustering, we used a dual surface labeling approach that allows detection of receptor–receptor interactions by TR-FRET. Because of the long-lived fluorescence of Lumi4-Tb, energy transfer in this assay results from both stable and transient protein–protein interactions, which increases when receptors are in closer proximity [38,39]. Using rat insulinoma INS-1 832/3 cells engineered by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) to delete endogenous GLP-1R [40] stably expressing human SNAP-GLP-1R (INS-1 832/3 GLP-1R −/− SNAP-GLP-1R cells), we detected a rapid increase in TR-FRET with exendin-4, which reached a maximum within 10 min (Fig 1D).…”

Section: Resultsmentioning

confidence: 99%

Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells

et al. 2019

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The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis. This process is dynamically regulated by agonist binding through palmitoylation of the GLP-1R at its carboxyl-terminal tail. Biased GLP-1R agonists and small molecule allosteric modulation both influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable.

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Section: Resultsmentioning

confidence: 99%

Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells

et al. 2019

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“…Non-specific FRET can arise from chance encounters occurring between fluorescent proteins attached to membrane proteins diffusing within a crowded membrane environment. This non-specific FRET must be exceeded to indicate specific physical interactions [38–42]. As defined previously [26], non-specific FRET was determined from control cells coexpressing YFP-tagged WT opsin and mTq2-tagged m2 muscarinic receptor, an unrelated GPCR (Fig.…”

Section: Resultsmentioning

confidence: 99%

Misfolded rhodopsin mutants display variable aggregation properties

Gragg

Park

2018

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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The largest class of rhodopsin mutations causing autosomal dominant retinitis pigmentosa (adRP) is mutations that lead to misfolding and aggregation of the receptor. The misfolding mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand the disease pathogenesis and evaluate therapeutic strategies. A Förster resonance energy transfer (FRET) method was utilized to directly assess the aggregation properties of misfolding rhodopsin mutants within the cell. Partial (P23H and P267L) and complete (G188R, H211P, and P267R) misfolding mutants were characterized to reveal variability in aggregation properties. The complete misfolding mutants all behaved similarly, forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and were unresponsive to treatment with the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. In the opsin form, the P23H mutant behaved similarly as the complete misfolding mutants. In contrast, the opsin form of the P267L mutant existed as both aggregates and oligomers when expressed alone and formed mostly oligomers with the wild-type receptor when coexpressed. The partial misfolding mutants both reacted similarly to the pharmacological chaperone 9-cis retinal, displaying improved folding and oligomerization when expressed alone but aggregating with wild-type receptor when coexpressed. The observed differences in aggregation properties and effect of 9-cis retinal predict different outcomes in disease pathophysiology and suggest that retinoid-based chaperones will be ineffective or even detrimental.

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“…FRET can occur from chance encounters of freely diffusing proteins that bring an acceptor and donor molecule within close proximity allowing for energy transfer even in the absence of physical interactions [43–45]. We refer to this type of FRET as non-specific FRET and that arising from physical interactions as specific FRET.…”

Section: Resultsmentioning

confidence: 99%

Wild-type opsin does not aggregate with a misfolded opsin mutant

Gragg

Kim

Howell

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Rhodopsin is the light receptor in photoreceptor cells that plays a central role in phototransduction and photoreceptor cell health. Mutations in rhodopsin are the leading cause of autosomal dominant retinitis pigmentosa (adRP), a retinal degenerative disease. A majority of mutations in rhodopsin cause misfolding and aggregation of the apoprotein opsin. The pathogenesis of adRP caused by misfolded opsin is unclear. It has been proposed that physical interactions between wild-type opsin and misfolded opsin mutants may underlie the autosomal dominant phenotype. To test whether or not wild-type opsin can form a complex with misfolded opsin mutants, we examined the interactions between wild-type opsin and opsin with a G188R mutation, a clinically identified mutation causing adRP. Förster resonance energy transfer (FRET) was utilized to monitor the interactions between fluorescently tagged opsins expressed in live cells. The FRET assay employed was able to discriminate between properly folded opsin oligomers and misfolded opsin aggregates. Wild-type opsin predominantly formed oligomers and only a minor population formed aggregates. Conversely, the G188R opsin mutant predominantly formed aggregates. When wild-type opsin and G188R opsin were coexpressed in cells, properly folded wild-type opsin did not aggregate with G188R opsin and was trafficked normally to the plasma membrane. Thus, the autosomal dominant phenotype in adRP caused by misfolded opsin mutants is not predicted to arise from physical interactions between wild-type opsin and misfolded opsin mutants.

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Lateral diffusion contributes to FRET from lanthanide-tagged membrane proteins

Cited by 11 publications

References 15 publications

Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells

Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells

Misfolded rhodopsin mutants display variable aggregation properties

Wild-type opsin does not aggregate with a misfolded opsin mutant

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