Endosomal binding kinetics of Eps15 and Hrs specifically regulate the degradation of RTKs

Abstract

Activation of EGF-R and PDGF-R triggers autophosphorylation and the recruitment of Eps15 and Hrs. These two endosomal proteins are important for specific receptor sorting. Hrs is recruiting ubiquitinated receptors to early endosomes to further facilitate degradation through the ESCRT complex. Upon receptor activation Hrs becomes phosphorylated and is relocated to the cytosol, important for receptor degradation. In this work we have studied the endosomal binding dynamics of Eps15 and Hrs upon EGF-R and PDGF-R stimulation. By analysing the fluorescence intensity on single endosomes after ligand stimulation we measured a time-specific decrease in the endosomal fluorescence level of Eps15-GFP and Hrs-YFP. Through FRAP experiments we could further register a specific change in the endosomal-membrane to cytosol binding properties of Eps15-GFP and Hrs-YFP. This specific change in membrane fractions proved to be a redistribution of the immobile fraction, which was not shown for the phosphorylation deficient mutants. We here describe a mechanism that can explain the previously observed relocation of Hrs from the endosomes to cytosol after EGF stimulation and show that Eps15 follows a similar mechanism. Moreover, this specific redistribution of the endosomal protein binding dynamics proved to be of major importance for receptor degradation.

De novo formation of an early endosome during Rab5 to Rab7 transition

“…We hypothesize that the Rab5 fractions on the newly formed endosome will be redistributed back to mobile fraction 80% and immobile fraction 20% in order to reactivate the fusion machinery. A redistribution of the immobile and mobile fraction of Hrs and Eps15 has previously been published to be important for EGFR degradation (Haugen et al, 2017). Similar redistribution of Rab5 was shown in this study which may indicate that a local redistribution of the mobile and immobile fraction on endosomes is a general mechanism to regulate endosomal maturation and progression.…”

“…showed us that EGFP-Rab5 has a mobile fraction of 22% and an immobile fraction of 78% (Figure 4), similar to previously observed values (Haugen et al, 2017). In order to compare the initial Rab5 detachment with the slower convergence maturation we had to specifically bleach the converging domains before the transfer to the newly formed Rab5 positive endosome.…”

“…Rab GTPases alternates between a membrane bound and a cytosolic state and we can measure the immobile and the mobile fraction present on the endosomes (Sprague and McNally, 2005). We have previously described that endosomal associated proteins can specifically alter the ratio between the mobile and the long lived immobile fraction both as a function of signaling and between interphase and mitosis (Bergeland et al, 2008;Haugen et al, 2017). As this is believed to reflect a functional state, we wanted to investigate whether these two fractions of Rab5 on the endosomal membrane varied through to the two detachment phases of Rab5.…”

“…The cytosolic tail of Ii has fusogenic properties that induce an enlargement of endosomes (Engering et al, 1998;Nordeng et al, 2002;Romagnoli et al, 1993;Stang and Bakke, 1997), without blocking maturation, or the transition from early to late endosome (Engering et al, 1998;Nordeng et al, 2002;Romagnoli et al, 1993;Stang and Bakke, 1997). This effect of Ii has been previously used to enlarge and study endosomal fusion/fission, coat binding kinetics, morphology, and maturation (Bergeland et al, 2008;Haugen et al, 2017;Landsverk et al, 2011;Skjeldal et al, 2012).…”

Invariant chain regulates endosomal fusion and maturation through the SNARE Vti1b

“…To test for SNAREs involved in Ii-mediated endosome enlargement, we silenced all individual SNAREs in the M1 fibroblast cell line, stably transfected with Ii under the control of a heavy-metal inducible promoter (M1 pMep4-Ii, (Nordeng et al 2002)). The M1 wild type cell line is negative for Ii and the MHCII proteins but the cell line has been employed extensively to study endosomal dynamics and antigen presentation by transfecting in immune genes (Roche et al 1993; Stang and Bakke 1997; Skjeldal et al 2012; Sand et al 2014; Haugen et al 2017). Ii transport to Ii positive endosomes was visualized by adding fluorescently tagged antibody recognizing the extracellular/luminal domain of Ii, and the size of Ii-positive endosomes was analysed by confocal microscopy (Figure 2A).…”