Proteolytic ectodomain shedding of membrane proteins in mammals-hardware, concepts, and recent developments

Abstract

Proteolytic removal of membrane protein ectodomains (ectodomain shedding) is a post-translational modification that controls levels and function of hundreds of membrane proteins. The contributing proteases, referred to as sheddases, act as important molecular switches in processes ranging from signaling to cell adhesion. When deregulated, ectodomain shedding is linked to pathologies such as inflammation and Alzheimer's disease. While proteases of the "a disintegrin and metalloprotease" (ADAM) and "beta-site APP cleaving enzyme" (BACE) families are widely considered as sheddases, in recent years a much broader range of proteases, including intramembrane and soluble proteases, were shown to catalyze similar cleavage reactions. This review demonstrates that shedding is a fundamental process in cell biology and discusses the current understanding of sheddases and their substrates, molecular mechanisms and cellular localizations, as well as physiological functions of protein ectodomain shedding. Moreover, we provide an operational definition of shedding and highlight recent conceptual advances in the field. While new developments in proteomics facilitate substrate discovery, we expect that shedding is not a rare exception, but rather the rule for many membrane proteins, and that many more interesting shedding functions await discovery.

Keywords: matrix metalloproteases; meprin β; pro‐protein convertases; rhomboids; signal peptide peptidase‐like.

Figure 1. Sheddases trigger the release of a wide range of proteins from the membrane

(A) Canonical sheddases cleave single‐pass TM membrane proteins in their luminal juxtamembrane region, thereby releasing ectodomains from their membrane‐integral domains. Ectodomain refers to that part of the protein that is found on the extracellular side of the membrane—in case that the protein localizes to the plasma membrane—or within the lumen of organelles of the secretory and endocytic pathway, which is topologically equivalent to the extracellular space. (B) GPI‐anchored proteins are separated from their lipid modification by cleavage within the C‐terminus of the protein. (C) Dual‐pass and polytopic membrane proteins (not shown) can be cleaved in loops and ectodomains (not shown). Neuregulin‐1 type III is cleaved at two sites in its loop domain, thereby releasing a bioactive peptide from its membrane anchors. (D) As a variation of canonical shedding, in regulated intramembrane proteolysis (RIP), the sheddase‐generated membrane‐integral fragment is further processed in the plane of the lipid bilayer, releasing an intracellular domain and a short extracellular peptide fragment. In this case, shedding is the first step of two subsequent proteolytic cleavages. (E) Non‐canonical sheddases cleave their substrate in or close to the TM domain without requiring any preceding cleavage. Depending on the site of cleavage, the intracellular fragment is released from the lipid bilayer or stays anchored by a slightly shortened TM domain.

Figure 2. Cellular localization of sheddases

Catalytically active canonical and non‐canonical sheddases not only localize to the cell surface but also to different subcellular compartments. The localization of selected canonical (red) and non‐canonical (green) sheddases is indicated.

Figure 3. Non‐canonical sheddases. One representative substrate per non‐canonical sheddase is given

(A) Rhomboid proteases cleave the TM domain of their substrates in the luminal membrane leaflet, thereby triggering release of the ectodomain. (B) SPP assembles with the rhomboid pseudoprotease Derlin1, and ERAD E3 ubiquitin ligases TRC8 and MARCH6 to form a proteolytic ERAD complex that recognizes membrane proteins without preceding cleavage. In a concerted action, fragments are released to both sides of the membrane and degraded by further components of the ERAD pathway. (C) SPPL3 cleaves glycan‐modifying enzymes at the luminal border of their TM domains, releasing the active site containing ectodomain. (D) Membrane proteins with large ectodomains need shedding to truncate their ectodomain before their C‐terminal fragment (CTF) can be further processed by γ‐secretase. In contrast, substrates with a naturally short ectodomain are directly shed by γ‐secretase leading to secretion of their entire ectodomains. Nicastrin (Nic) serves as a molecular ruler accepting only membrane proteins with a short ectodomain.

Figure 4. Functional consequences of shedding

(A) By proteolytic processing of TM proteins that display a physiological function, like cell adhesion or receptor‐mediated signaling, sheddases terminate these functions. (B) Shedding generates biologically active signaling molecules from membrane‐anchored precursors, e.g., cytokines or growth factors that act on neighboring or far distant cells. (C) As part of regulated intramembrane proteolysis, sheddases induce a proteolytic cascade ultimately activating signaling molecules, like the Notch intracellular domain, that act within the same cell. Shedding may be ligand‐induced and the ligand may even be a membrane‐anchored protein itself, as in case of Notch.