Transforming growth factor-alpha and beta-amyloid precursor protein share a secretory mechanism.

Abstract

Abstract. Cleavage and release of membrane protein ectodomains, a regulated process that affects many cell surface proteins, remains largely uncharacterized. To investigate whether cell surface proteins are cleaved through a shared mechanism or through multiple independent mechanisms, we mutagenized Chinese hamster ovary (CHO) cells and selected clones that were unable to cleave membrane-anchored transforming growth factor o~ (TGF-a). The defect in TGF-a cleavage in these clones is most apparent upon cell treatment with the protein kinase C (PKC) activator PMA, which stimulates TGF-ct cleavage in wild-type cells. The mutant clones do not have defects in TFG-ct expression, transport to the cell surface or turnover. Concomitant with the loss of TGF-ot cleavage, these clones have lost the ability to cleave many structurally unrelated membrane proteins in response to PMA. These proteins include ~-amyloid precursor protein (/3-APP), whose cleavage into a secreted form avoids conversion into the amyloidogenic peptide A/~, and a group of cell surface proteins whose release into the medium is stimulated by PMA in wild type CHO cells but not in mutants. The mutations prevent cleavage by PKC-dependent as well as PKC-independent mechanisms, and thus affect an essential component that functions downstream of these various signaling mechanisms. We propose that regulated cleavage and secretion of membrane protein ectodomains is mediated by a common system whose components respond to multiple activators and act on susceptible proteins of diverse structure and function.

Characterisation of endothelin converting enzyme‐1 shedding from endothelial cells

“…All MALDI spectra at time = 0 min confirmed the presence of BigET [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] , but neither the C-terminal cleavage product BigET [22][23][24][25][26][27][28][29][30][31][32][33][34] (MW 1380 Da), nor the N-terminal cleavage product BigET 18-21 (MW 545 Da) were detected (data not shown). BigET [22][23][24][25][26][27][28][29][30][31][32][33][34] was detected in the mixture containing media alone, and in the presence of thiorphan after 2 and 5 h, respectively ( Fig. 3a and b).…”

“…Conditioned media from HUVECs (60 lL) was added to the specific substrate BigET [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] ; (MW 1907 Da; DIIWVNTPEHVVPYGLG; Auspep, Vic, Australia), or the natural substrate BigET (MW 4283 Da) giving a final concentration of 33 or 66 lg/mL, respectively, in ECE-1 assay buffer. The reaction mixture was incubated at 37°C.…”

“…Proteolytic shedding of ECE-1 was measured by monitoring the cleavage of quenched fluorescent substrate (QFS) in conditioned media. A mass spectrometry approach involving BigET [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] , was used to confirm for the first time that ECE-1, endogenously expressed by human umbilical vein endothelial cells undergoes ectodomain shedding. Our data also suggest that ADAM family of metalloproteinases or MMPs are not involved in the ECE-1 ectodomain cleavage.…”

Extracellular Proteases in Atherosclerosis and Restenosis

“…One of the best-studied shedding processes is the cytokine TNF-␣, being released from its membrane-bound precursor by the ADAM-17 member (also known as TNF-␣ converting enzyme). 76 Other examples important to the vasculature are the shedding of TGF-␣, 77 FGF receptor-1, 78 IL-6 receptor, 79 macrophage colony-stimulator factor, 79 angiotensinconverting enzyme, 80 L-selectin, 81 and vascular cell adhesion molecule. 82 Interestingly, many nonsteroidal antiinflammatory drugs such as aspirin exert anti-inflammatory properties by inducing the L-selectin shedding in neutrophils 83 in addition to inhibiting prostaglandin synthesis through the cyclooxygenase blockade.…”

Inside-out Regulation of Ectodomain Cleavage of Cluster-of-Differentiation-44 (CD44) and of Neuregulin-1 Requires Substrate Dimerization

“…intracellular calcium influx and the release of diacylglycerol, which activates protein kinase C (13)(14)(15). Thus, intracellular signaling pathways regulate a process (known as substrate protease accessibility) that occurs outside of the plasma membrane.…”