Receptor Heterodimerization Modulates Endocytosis through Collaborative and Competitive Mechanisms

Abstract

Recruitment of receptors into clathrin-coated structures is essential to signal transduction and nutrient uptake. Among the many receptors involved in these processes, a significant fraction forms dimers. Dimerization of identical partners has generally been thought to promote receptor recruitment for uptake because of increased affinity of the dimer for the endocytic machinery. But what happens when receptors with substantially different affinities for the endocytic machinery come together to form a heterodimer? Evidence from diverse receptor classes, including G-protein-coupled receptors and receptor tyrosine kinases, suggests that heterodimerization with a strongly recruited receptor can drive significant recruitment of a receptor that lacks direct interactions with the endocytic machinery. However, a systematic biophysical understanding of this effect has yet to be established. Motivated by the potential of such events to influence cell signaling, here, we investigate the impact of receptor heterodimerization on endocytic recruitment using a family of engineered model receptors. As expected, we find that dimerization of a weakly recruited receptor with a strongly recruited receptor promotes incorporation of the weakly recruited receptor to endocytic structures. However, the effectiveness of this collaborative mechanism depends heavily on the relative strengths of endocytic recruitment of the two receptors that make up the dimer. Specifically, as the strength of endocytic recruitment of the weakly recruited receptor approaches that of the strongly recruited receptor, monomers of each receptor compete with heterodimers for space within endocytic structures. In this regime, the presence of the strongly recruited receptor drives a reduction in incorporation of the weakly recruited receptor into clathrin-coated structures. Similarly, as the strength of the dimer bond between the two receptors is progressively weakened, competition begins to dominate over collaboration. Collectively, these results demonstrate that the impact of receptor heterodimerization on endocytic recruitment is controlled by a delicate balance between collaborative and competitive mechanisms.

Figure 1

Binding to a strongly recruited receptor promotes endocytic recruitment of a weakly recruited receptor. (A–C) Cartoon schematics show the architecture of the YTRF model receptor (A), the CTRD model receptor (B), and the YTRF helper receptor (C). (D–F) Spinning disk confocal images show the plasma membrane of RPE cells stably expressing the clathrin light chain (CLC) tagged with BFP and transiently expressing the YTRF model receptor, M_YTRF (D), the CTRD model receptor, M_CTRD (E), and the YTRF helper receptor, H_YTRF (F). The box in the top image represents the location of the inset and the dashed circles in the insets highlight the diffraction-limited puncta colocalized between receptors and clathrin channels. It is important to note that these structures are much smaller than the circles drawn around them. (G) A cartoon schematic shows that M_CTRD and H_YTRF are incorporated into CCSs as heterodimers, leading to increased endocytic recruitment of M_CTRD. (H) As in (D)–(F), a confocal image shows M_CTRD and H_YTRF coexpressed in an RPE cell. All scale bars in example images, 5 μm; scale bars in insets, 1 μm. To see this figure in color, go online.

Figure 2

Equilibrium partitioning of receptors between clathrin-coated structures (CCSs) and the plasma membrane. (A and B) Lifetime cohorts of tracked CCS intensities in receptor (GFP; dashed line) and clathrin light-chain (CLC) (mCherry; solid line) channels are shown. 4500 CCSs from 15 total cells expressing M_YTRF were analyzed in (A), and 7754 CCSs from 16 total cells expressing M_CTRD were analyzed in (B). Notably, the green curves are noisier than the red curves, likely because of the lower signal/noise ratio in this channel. (C) The lifetime distributions of CCSs from cells expressing no model cargo, M_CTRD, or M_YTRF are shown. The average lifetime of all mCherry-CLC positive tracks across all lifetimes was 52 ± 2 s 95% confidence interval (CI) for cells with no cargo expression, 51 ± 1 s 95% CI for cells expressing M_CTRD, and 58 ± 1 s 95% CI for cells expressing M_YTRF (N = 1648, 7754, and 4500 CCSs, respectively). (D and E) Image sequences depict the recovery of the bleached plasma membrane for cells expressing either M_CTRD alone (D) or both M_CTRD and H_YTRF (E). The dashed line shows the edges of the photobleached area. GFP and RFP contrast settings are displayed in arbitrary units directly above each image sequence. Dashed circles in the BFP channel images show the recovered CCSs. All scale bars, 5 μm. (F and G) Normalized recovery curves are shown as mean (circles) ± standard error of the mean (shaded area). The recovery half time (t_1/2), extracted from the exponential fitting (black dashed line), is shown as mean with 95% CI. N = 7 and 9 cells for (F) and (G), respectively. To see this figure in color, go online.

Figure 3

Reduced endocytic recruitment of the helper receptor results in reduced recruitment of the weakly recruited model receptor. (A) A cartoon schematic shows the CTRD model receptor bound to the YXXΦ mutants of the helper receptors. (B) <n> versus C_mem plot is shown. The average number of receptors per CCS is shown as a function of the receptor density on the surrounding plasma membrane. Each point is shown as the average of 250 puncta binned by receptor density on membrane surface ± standard error of the mean. Dashed lines indicate model predictions using Eq. 1. This plot shows 61 points representing 15,202 puncta for H_YTRF and 133 points representing 33,290 puncta for M_CTRD. (C) <n> versus C_mem plot in arbitrary units (AU) is shown. The average concentration of the helper receptor was held constant at ∼8000 AU per μm² (Fig. S7). This plot shows 61 points representing 15,202 puncta for H_YTRF, 78 points representing 19,426 puncta for M_CTRD + H_YTRF, 60 points representing 14,933 puncta for M_CTRD + H_YARI, 75 points representing 18,722 puncta for M_CTRD + H_CTRF, and 133 points representing 33,290 puncta for M_CTRD. (D and E) Spinning disk confocal images show the plasma membrane of BFP-CLC RPE cells transiently expressing M_CTRD with either H_YARI (D) or H_CTRF (E). Scale bars in example images, 5 μm; scale bars in insets, 1 μm. (F) Bar plot summarizing the effective dissociation constants is shown as the average, with error bars indicating the 95% CI. To see this figure in color, go online.

Figure 4

Increased endocytic recruitment of the model receptor leads to competition with the helper receptor. (A) A cartoon schematic shows the YTRF helper receptor bound to the YXXΦ mutants of the model receptors. (B–D) <n> versus C_mem plot in arbitrary units (AU) as in Fig. 3C is shown. The estimated Kd_eff for (B) and (C) are summarized in inset bar plots with 95% CI. For (B) and (C), the average concentration of the helper or competitor receptor was held constant at ∼8000 AU per μm² (Fig. S8). For comparison, the H_YTRF curve is replotted here from Fig. 3C. The plot in (B) shows 61 points representing 15,202 puncta for M_YTRF, 71 points representing 17,713 puncta for M_CTRF + H_YTRF, and 144 points representing 36,099 puncta for M_CTRF. The plot in (C) shows 150 points representing 37,572 puncta for M_YARI, 93 points representing 23,347 puncta for M_YARI + H_YTRF, and 136 point representing 33,982 puncta for M_YARI + C_YTRF. The plot in (D) shows 150 points representing 37,572 puncta for M_YARI, 34 points representing 8566 puncta for M_YARI + H_YTRF – low expression, 31 points representing 7710 puncta for M_YARI + H_YTRF – medium expression, and 28 points representing 7071 puncta for M_YARI + H_YTRF – high expression. (E) Cartoon schematics show relative endocytic recruitment of M_YARI when 1) expressed alone, 2) with H_YTRF, or 3) with C_YTRF. (F) Cumulative <n> versus cumulative C_mem plot in AU is shown. Each point is shown as the average of 250 puncta binned by cumulative receptor density on membrane surface ± standard error of the mean. This plot shows 45 points representing 11,374 puncta for M_YARI + H_YTRF and 65 points representing 16,248 puncta for M_YARI + C_YTRF. The bar graph shows the average cumulative <n> in AU over the entire range of cumulative C_mem on the plot. Error bars represent standard error of the mean. Asterisks represent statistically significant differences in two-tailed t-test with p < 0.05. To see this figure in color, go online.

Figure 5

Reduced binding between the model and helper receptors reduces endocytic recruitment of the weakly recruited model receptor. (A) A cartoon schematic shows M_CTRD bound to H_YTRF. The zoomed-in image shows the key amino acids in the binding interface between GFP nanobody and GFP. (B) Cartoon schematics show the relative endocytic recruitment of M_CTRD in the presence of H_YTRF with mutations on the GFP nanobody domain, either R35A (top) or R35A and E103A (bottom). (C and D) Spinning disk confocal images show the plasma membrane of BFP-CLC RPE cells transiently expressing M_CTRD with either H_YTRF R35A (C) or H_YTRF R35A E103A (D). Scale bars in example images, 5 μm; scale bars in insets, 1 μm. (E and F) <n> versus C_mem plot in arbitrary units (AU) as in Fig. 3C is shown. The average concentration of the helper or competitor receptor was held constant at ∼5500 AU per μm² (Fig. S11). The best fit Kd_eff values are summarized in (F) with 95% CI. For comparison, curves for M_CTRD and M_CTRD + H_YTRF are replotted here from Fig. 3C. This plot shows 74 points representing 18,579 puncta for M_CTRD + H_YTRF, 114 points presenting 28,495 puncta for M_CTRD + H_YTRF R35A, 143 points representing 35,764 puncta for M_CTRD + H_YTRF R35A E103A, 133 points representing 33,290 puncta for M_CTRD, and 151 points representing 37,679 puncta for M_CTRD + C_YTRF. (G) Cumulative <n> versus cumulative C_mem plot in AU as in Fig. 4F is shown. This plot shows 62 points representing 15,503 puncta for M_CTRD + H_YTRF, 104 points representing 26,116 puncta for M_CTRD + H_YTRF R35A, 108 points representing 26,976 puncta for M_CTRD + H_YTRF R35A E103A, and 90 points representing 22,660 puncta for M_CTRD + C_YTRF. The bar graph shows the average cumulative <n> in AU over the entire range of cumulative C_mem on the plot. Error bars represent standard error of the mean. Asterisks represent statistically significant differences in two-tailed t-test with p < 0.05. To see this figure in color, go online.