Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging - PubMed (original) (raw)

Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging

Malte Renz et al. Proc Natl Acad Sci U S A. 2012.

Abstract

The stoichiometry and composition of membrane protein receptors are critical to their function. However, the inability to assess receptor subunit stoichiometry in situ has hampered efforts to relate receptor structures to functional states. Here, we address this problem for the asialoglycoprotein receptor using ensemble FRET imaging, analytical modeling, and single-molecule counting with photoactivated localization microscopy (PALM). We show that the two subunits of asialoglycoprotein receptor [rat hepatic lectin 1 (RHL1) and RHL2] can assemble into both homo- and hetero-oligomeric complexes, displaying three forms with distinct ligand specificities that coexist on the plasma membrane: higher-order homo-oligomers of RHL1, higher-order hetero-oligomers of RHL1 and RHL2 with two-to-one stoichiometry, and the homo-dimer RHL2 with little tendency to further homo-oligomerize. Levels of these complexes can be modulated in the plasma membrane by exogenous ligands. Thus, even a simple two-subunit receptor can exhibit remarkable plasticity in structure, and consequently function, underscoring the importance of deciphering oligomerization in single cells at the single-molecule level.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Differential asialoglycoprotein receptor subunit assembly dictates ligand specificity. NRK cells expressing different combinations of GFP-RHL1, GFP-RHL2, and/or Cherry-RHL2 were examined for their ability to bind Alexa647-labeled ASF or Alexa647-LTF. Green, red, and far-red channels are displayed. Mean fluorescence intensity (F) was calculated by integrating over the pixels corresponding to the plasma membrane. (A) Cells expressing either RHL1 or RHL2 alone did not bind ASF. (B) Coexpression of RHL1 and RHL2 resulted in efficient ASF binding. (C) RHL1 expression alone resulted in efficient LTF binding. (D) RHL2 expression alone did not result in efficient LTF binding. (E) Expressing both subunits in comparable amounts did not lead to LTF binding. (F) RHL1 expression at high levels relative to RHL2 led to detectable LTF binding. (Scale bar: 10 μm.)

Fig. 2.

Fig. 2.

Ensemble FRET analyses show distinct homo-oligomerization of RHL1 and RHL2 driven by different molecular motifs. The cartoons depict the studied subunits or their mutant forms. The images display color-coded FRET values in each pixel. The color-coded scale bar is for normalized mean FRET efficiencies. (Scale bar: A–C, 10 μm.) In the graphs, mean normalized FRET efficiencies integrated over the pixels corresponding to the plasma membrane were plotted vs. the acceptor-to-donor ratio. (A) Homo-oligomerization of RHL1. Plotting the FRET efficiency vs. the acceptor-to-donor ratio revealed that the detected FRET values for RHL1 homo-association rose well beyond the positive control and did not plateau, indicating that RHL1 homo-associates into large clusters. (B) Deletion of RHL1’s stalk domain (RHL1Δc) and all external domains (RHL1TM) led to a normalized FRET efficiency of about 25% between WT and mutant RHL1 forms and among the mutants. This finding suggests that the major driving force for RHL1 homo-oligomerization is the stalk domain. (C) Homo-oligomerization of RHL2. RHL2 forms homo-dimers and not higher-order oligomers. Deletion of all external domains of RHL2 did not change the normalized FRET efficiencies. Thus, RHL2 homo-oligomerization is mainly determined by transmembrane and intracellular domains. (D) Self-association of RHL1 decreases in the presence of RHL2. RHL1 homo-association is shown side by side in the presence and absence of PA-Cherry–labeled RHL2. FRET between RHL1 molecules was measured before activation of PA-Cherry. (E) Self-association of RHL2 is unchanged in the presence of RHL1. RHL2 self-association is shown side by side in the presence and absence of PA-Cherry–labeled RHL1. FRET between RHL2 molecules was measured before activation of PA-Cherry.

Fig. 3.

Fig. 3.

Ensemble FRET analyses reveal an asymmetric receptor subunit assembly of the asialoglycoprotein receptor. (A) FRET efficiencies between GFP-labeled RHL1 (donor) and Cherry-labeled RHL2 (acceptor). The detected FRET curve exhibits a steep increase followed by an early plateau. The cartoons in A and B depict the studied RHL1 and RHL2 labeled with donor or acceptor, respectively. The images display the color-coded FRET values in each pixel. Mean FRET efficiency integrated over the pixels showing the plasma membrane was plotted vs. the acceptor-to-donor ratio. The color-coded scale bar is for normalized mean FRET efficiencies. (Scale bar: A and B, 10 μm.) (B) FRET efficiencies of Cherry-labeled RHL1 and GFP-labeled RHL2. The FRET curve exhibits an almost linear increase and plateaus only at a high acceptor-to-donor ratio. (C) Schematic explanation for the detected nonidentical FRET curves of RHL1/RHL2 hetero-oligomerization. Insets show the simplest explanation of an asymmetric receptor subunit hetero-oligomerization: a stoichiometric distribution of two RHL1 dimers to one RHL2 dimer in a receptor complex. Hypothesized FRET curves consistent with this explanation that match the experimental data in A and B are shown in the graphs. (D) Analytical model for the schematic explanation above (

SI Experimental Procedures

). The model calculations showed that the experimental data of A and B (shown as gray and black dots, respectively) can be fit (gray and black lines, respectively) to a receptor subunit stoichiometry of 2:1 (RHL1:RHL2) and ruled out a symmetric receptor subunit hetero-oligomerization.

Fig. 4.

Fig. 4.

Single-molecule counting PALM visualizes an asymmetric receptor subunit assembly of asialoglycoprotein receptor on the single-molecule level. (A) Single-molecule counting PALM was capable of stably reproducing a specific red-to-green ratio in different cells expressing the vesicular stomatitis virus glycoprotein-PA-GFP-PA-Cherry double construct in different absolute amounts. (B) Strategy for determining the relative detection efficiency of PA-GFP and PA-Cherry using a calibration probe. (C) Applying single-molecule counting PALM to cells expressing vesicular stomatitis virus glycoprotein constructs containing PA-GFP and PA-Cherry in differing amounts permits assessment of the correct stoichiometry of the constructs. Three constructs having the following ratios of PA-Cherry and PA-GFP were assessed: 1 PA-Cherry:2 PA-GFP; 1 PA-Cherry:1 PA-GFP; and 2 PA-Cherry:1 PA-GFP. The red-to-green ratio in each cell expressing the given constructs was determined and normalized to the relative detection efficiency. (D) Coexpression of RHL1 labeled with PA-GFP and PA-Cherry. Applying single-molecule counting PALM, we detected 40 molecules/μm2 and a relative red-to-green ratio of 0.85 in a membrane area of 36 μm2. The relative red-to-green ratio within an RHL1 homo-oligomeric cluster was 0.88 ± 0.1. (E) Coexpression of PA-GFP-RHL1 and PA-Cherry-RHL2. Applying single-molecule counting PALM, we detected 40 molecules/μm2 and a relative red-to-green ratio of 0.81 in a membrane area of 36 μm2. The relative red-to-green ratio of 0.43 ± 0.09 within a hetero-oligomeric cluster, however, deviated significantly from the relative overall expression. RHL1 was more abundant inside the clusters, indicating an asymmetric receptor assembly that approximates a stoichiometry of two RHL1 to every one RHL2 in a cluster. (F) Plotting the detected number of red and green molecules inside the homo- and hetero-oligomeric clusters can be fit to straight lines with a slope of 0.88 ± 0.15 for the RHL1 homo-oligomers as displayed in D and 0.45 ± 0.16 for the RHL1/RHL2 hetero-oligomers as shown in E. The slope of the line fit gives an estimate for the average stoichiometry within a cluster and coincides with the red-to-green ratio within the homo- and hetero-oligomeric clusters, respectively.

Fig. 5.

Fig. 5.

Assembly of asialoglycoprotein receptor into different homo- and hetero-oligomeric states with distinct ligand specificities defines receptor plasticity and can be modulated further by exogenous ligands. (A) The assembly of the asialoglycoprotein receptor on the plasma membrane can be pictured as a modular system. Because of its high tendency to homo-associate, RHL1 will form homo-oligomers that can bind lactoferrin. When RHL2 homo-dimers are present at the plasma membrane, they associate with RHL1 mediated through the stalk domain and weaken the tendency of RHL1 to self-associate. RHL1 and RHL2 form hetero-oligomers comprising receptor cores of two RHL1 homo-dimers and one RHL2 homo-dimer. Both the homo-oligomeric complexes of RHL1 and the hetero-oligomeric complexes can further associate to higher-order oligomers that, in the case of the hetero-oligomer, preserve a 2:1 stoichiometry of RHL1:RHL2. (B) Differential oligomerization driven by different molecular motifs and different subunit abundances leads to the coexistence of different oligomers with distinct ligand specificities at the plasma membrane. (C) The steady-state equilibrium of the distinct coexistent homo- and hetero-oligomeric receptor states can be shifted by exogenous ligand. When asialofetuin is added, it binds to the hetero-oligomeric complexes and leads to their internalization, leaving behind RHL1 homo-oligomers. Because RHL1 is labeled with GFP, donor-only complexes will be enriched at the plasma membrane, which results in a decrease in detected normalized FRET efficiencies of RHL1 labeled with GFP and RHL2 labeled with Cherry. The addition of lactoferrin, however, leads to the internalization of donor-only RHL1 homo-oligomers, leaving behind the hetero-oligomeric complexes and thus results in an increase in detected FRET efficiencies.

Fig. P1.

Fig. P1.

(A) Ensemble FRET imaging combined with mathematical modeling helps dissect receptor subunit assembly and stoichiometry. Shown are representative images of fluorescence in the donor, acceptor, and FRET channel, data analysis of normalized FRET efficiency values in each image pixel, and modeling of receptor subunit hetero-oligomerization. (B) Single-molecule counting PALM directly visualizes receptor subunit assembly and stoichiometry at the single-molecule level in situ. Shown are sequential single-molecule image acquisitions in green and red channels, standardization of single-molecule counting for assessing relative expression, and stoichiometries by counting calibrators consisting of spectrally distinct photoactivatable fluorescent proteins. (C) The combined use of these spectroscopic methods results in a model of different coexistent receptor oligomers determined by the tendency of receptor subunits to form oligomers, the relative subunit abundances, and the presented ligands. The model defines plasticity of the two-subunit receptor system.

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