Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis

Thank you for submitting your article "Endogenous pH-sensitive EGF receptor and its application in high-throughput analysis of clathrin-mediated endocytosis" for consideration by eLife. Your article has been favorably reviewed by two peer reviewers, and the evaluation has been overseen by John Kuriyan as the Reviewing Editor and Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: H Steven Wiley (Reviewer #1).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. You will see that no new experiments are asked for, and so we do not anticipate any difficulties in revising the paper.

Summary:

This is a nice technical advance that describes a novel approach for high-throughput screening of EGFR endocytosis. Larsen et al. report development and characterization of an assay to measure the rate of clathrin-mediated endocytosis (CME) of EGFR using HeLa cells in which CRISPR has been used to replace endogenous EGFR with a form of EGFR that can be labeled with a pH-sensitive fluorophore. The controls show that this tagging (FAP tagging) does not impact the internalization or physiological activity of the EGFR. In addition, they show that the sensitivity of the ratiometric technique is sufficient to allow assessment of physiological levels of ligand, and thus can be used to follow relevant mechanisms that regulate trafficking under physiological conditions.

This assay closely resembles a similar assay developed using transfection of a labeled β2-adrenergic receptor in HEK293, which was published by the Bruchez group in Biochemistry in 2018. The authors employ this assay in an siRNA screen of 240 phosphatases to identify phosphatases involved in CME of EGFR. At least three phosphotidyl-inositide phosphatases along with several phosphatases previously implicated in dephosphorylation of EGFR were identified in this screen. The precise roles of these phosphatases in CME of EGFR will be the subject of future studies.

On balance, the FAP-tagging approach seems ideally suited for high-throughput screening experiments because of its ability to use a single emission wavelength for the readout, the uniformity of the labeling, the relatively high S/N ratio that permits the use of low ligand concentrations and the ability to follow both empty and occupied receptors. The clean and consistent results obtained with both clathrin and dynamic siRNA pools is impressive (Figure 4). For screens of molecules that perturb the endocytosis of the EGFR (or other surface-associated proteins that can be tagged), this is certainly a powerful approach. It also shows the power of CRISPR-mediated tagging approaches and is likely to inspire the application of this technology to similar problems in biology.

Essential revisions:

Please discuss the following issues, to place the work in better context, noting the comments made by the reviewers below.

Please discuss the earlier work by others on the β2-adrenergic receptor more clearly, showing that the present work is an extension of work that had already developed the basic technology. We recognize that the advance here is to tag the endogenous receptor.

Although this is clearly a very powerful technological advance, there needs to be more discussion of both previous approaches and the advantages and disadvantages of the FAP-tagging approach as compared to those approaches. In particular, the need to genetically modify a cell line prior to use is a substantial disadvantage.

Also, the paper implies that endocytosis in their current cell line of choice (HeLa) is the same as all other cells, which the authors themselves have shown not to be true. For example, constitutive internalization of EGFR in mammary epithelial cells is quite distinct from most other cell lines, such as HeLa and fibroblasts (Burke and Wiley, 1999). EGFR endocytosis in mouse fibroblasts is also quite distinct from what is observed in HeLa and endothelial cells (Jiang et al., 2003). Thus, the approach outlined here would require tagging a variety of different cell lines to fully explore endocytic mechanisms and screen for regulators, which could be technically difficult.

It also has the disadvantage of being most useful for cells that express relatively low levels of receptors so that the signal from low levels of internalized receptors is not swamped out by high levels of receptors remaining on the cell surface.

Conversely, their approach has many advantages as compared to commonly used techniques. In particular, virtually all described methods for examining endocytosis of the EGFR are restricted to ligand-occupied receptors that behave dramatically different than empty receptors. In this respect, the technique is similar to the antibody-tagging technique (e.g. Burke, Schooler and Wiley, 2001), which has its own set of disadvantages, such as either blocking receptor occupancy (e.g. 225 mAb) or restricting the ligand which can be used (e.g. 13A9 mAb). However, radiolabeled antibodies have the advantage of yielding extremely high S/N ratios, thus also permitting evaluation of very low levels of receptor occupancy, even in cells with high levels of receptors.