Dual clathrin and adhesion signaling systems regulate growth factor receptor activation

Flat clathrin lattices sustain growth factor signaling via integrins

 

Background

Clathrin is an endocytic protein that drives the internalization of ligand-engaged (activated) cell-surface receptors via clathrin-coated pits, but it also regulates the assembly of large, sheet-like structures called flat clathrin lattices (FCLs) underneath the plasma membrane. In contrast to pits, FCLs are static and long-lived and their assembly is thought to result from ‘frustrated endocytosis’, with budding pits being observed only at their periphery [1]. However, a more active role for FCLs has recently become apparent in cell signaling, proliferation, and mechanotransduction [2,3]. Intriguingly, FCLs are immobilized by binding to the extracellular matrix via a transmembrane cell adhesion receptor of the integrin family, integrin αvβ5, and they are therefore increasingly recognized as a novel type of adhesion complex (also known as ‘reticular adhesions’) [4,5]. While these studies indicate that FCLs may constitute localized platforms for the integration of adhesion, growth factor signaling, and endocytosis, it is to date unknown how such integration of signals is regulated.

 

Key findings

In this preprint, Alfonzo-Mendez et al. identify a signaling axis that regulates FCL assembly during epidermal growth factor (EGF) stimulation, and uncover a novel layer of crosstalk between growth factor receptors and adhesion proteins. The authors mechanically ‘unroofed’ cells to directly visualize clathrin-containing structures at the cytoplasmic face of the plasma membrane using platinum replica transmission electron microscopy (PREM). Furthermore, they assessed the localization of signaling proteins at thousands of individual clathrin-coated structures in an unbiased manner by total internal reflection fluorescence microscopy (TIRFM). In this way, the authors show that EGF stimulates the generation and expansion of FCLs, which is prevented by pharmacological inhibition of the EGF receptor (EGFR), the tyrosine kinase Src, or integrin αvβ5. EGF also promotes the recruitment of activated EGFR, Grb2, and integrin αvβ5 into FCLs. Like many other β-integrin cytoplasmic tails, β5 contains two NPxY motifs, which recruit signaling and trafficking proteins, and whose tyrosines can be phosphorylated. The authors show that these tyrosines in β5 are phosphorylated by Src, which is required for αvβ5 to localize to FCLs. Interestingly, Src itself is excluded from FCLs after EGF addition, suggesting that Src leaves these structures after phosphorylating the β5 cytotail. While it remains to be determined how the NPxY phosphorylations affect αvβ5 function, it is clear that ligand binding by αvβ5 is crucial for growth factor signaling in FCLs, as the antagonist cilengitide blocks EGF-dependent FCL formation, but also EGFR activation, internalization, and the recruitment of Grb2. Thus, FCLs act as signaling platforms at the plasma membrane that localize and sustain growth factor signaling via integrins, and the crosstalk between growth factor and adhesion receptor signaling at FCLs is mediated by Src.

Figure. Image acquired by PREM of clathrin domes, spheres, and FCLs after EGF addition.

 

What I like

It has been well-established that integrins and growth factor receptors mutually affect each other’s signaling and trafficking. Here, the authors used elegant methods to visualize the EGF-mediated assembly of FCLs, and the co-localization of their components with clathrin. Their data reveal how the highly specialized integrin αvβ5 drives localized and sustained EGF signaling, and uncover a novel layer of crosstalk between growth factor and cell adhesion.

 

Future directions

It will be very interesting to determine whether other receptor tyrosine kinases, such as the receptors for vascular endothelial growth factor or fibroblast growth factor, also regulate FCL assembly, and if they localize and signal from these structures in a similar way as EGFR. Furthermore, how the behavior of integrin αvβ5 is controlled in and out of FCLs requires further investigation. While Src can also phosphorylate other integrins, those integrins are not found in FCLs but instead localize to focal adhesions, suggesting that other factors besides Src contribute to the unique role of αvβ5 in the formation and function of FCLs. Finally, the physiological role of FCLs remains to be determined. To date they have only been observed in muscle in vivo, but they are likely to be important in other cell types as well.

 

References

• Sochacki KA, et al. (2017) Endocytic proteins are partitioned at the edge of the clathrin lattice in mammalian cells.  Nat. Cell Biol.19, 352-361
• Leyton-Puig D, et al. (2017) Flat clathrin lattices are dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling of specific receptors.  Nat. Commun.8, 1-14
• Baschieri F, et al. (2018) Frustrated endocytosis controls contractility-independent mechanotransduction at clathrin-coated structures.  Nat. Commun.9, 3825
• Zuidema A, et al. (2018) Mechanisms of integrin αVβ5 clustering in flat clathrin lattices.  J. Cell Sci.131, jcs221317
• Lock J, et al. (2018) Reticular adhesions are a distinct class of cell-matrix adhesions that mediate attachment during mitosis.  J. Cell Biol. 20, 1290-130

 

 

 

 

 

doi: https://doi.org/10.1242/prelights.26226

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