Consider a cell surface receptor that upon ligand binding, becomes mono-ubiquiti

Question

Consider a cell surface receptor that upon ligand binding, becomes mono-ubiquitinated, undergoes endocytosis, and finally degradation via the multivesicular body pathway. You probe the system using siRNA to deplete cells of various components and observe the following experimental outcomes. Please rationalize each experimental outcome using the mechanisms we learned about in lecture A. Knocking down either the the relevant ubiquitin ligase or Hrs or tsg101 causes a lack of degradation of the receptor. Confusingly, the receptor continues to undergo rapid endocytosis upon stimulation, but steady-state surface levels of the receptor are only negligibly reduced. B. Knocking down various other components of ESCRT1, ESCRT2, or ESCRT3 results in permanent removal of receptor from the plasma membrane. However, it still does not get degraded. Instead it accumulates intracellularly. C) Knocking down vps4 causes a complex phenotype. The receptor undergoes endocytosis upon stimulation as normal. Initially there is some receptor degradation indicated by a slight drop in the total cellular receptor level. However after this the total receptor population is stable and oddly, as in part A at steady state almost 100% of the receptor population is found on the cell surface

Explanation / Answer

A. A set of multiprotein complexes associated with the endosome membrane are collectively referred to as ESCRT or endosomal sorting complex required for transport- ESCRT 0, ESCRT I, ESCRT II, ESCRT III. These complexes mediate the ubiquitin directed sorting into multivesicular bodies (MVB).

HRS-STAM or hepatocyte growth factor regulated tyrosine kinase substrate – signal transducing adaptor molecule complex is one of the main components of ESCRT 0. This complex interacts directly with ubiquitylated receptors and facilitates the recruitment of ESCRT I to the MVB membrane. The ESCRTI and ESCRT III complexes are required to recruit enzymes for deubiquitylation of receptors and then they are packaged into ILVs (intraluminal vesicles) of multivesicular bodies or MVBs.

Thus the level of surface receptors is only negligibly reduced.

B. After internalization the signaling receptors are sorted to recycling. There is endosomal degradation pathway in the same manner as other endocytic molecules. It is now established that specific sequence motifs control the recycling of receptors.

The endosomal sorting of receptors targets them to late endosomes and lysosomes for degradation. Thus ligand induced ubiquitylation plays a key role in the downregulation of the receptors. These receptors are accumulated intracellularly.

C. The GTP binding alpha subunit or Gpa1 mediates with MAPK signaling from the plasma membrane. The Gpa 1 translocates to endosomes following GTP hydrolysis and can bind to the endosome associated protein Vps15.This protein is structurally homologous to G protein beta subunits associated with the P13K Vps34. GTP bound Gpa1 activates Vps34 Kinase activity. This subsequently leads to the activation of MAPKs and other signaling cascades.

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