Two water channel homologs were cloned recently from rat kidney, mercurial-insensitive water channel (MIWC) and glycerol intrinsic protein (GLIP). Polyclonal antibodies were raised against synthetic C-terminal peptides and purified by affinity chromatography. MIWC (Pierce). MIWC antibodies were raised in rabbits and GLIP antibodies were raised in mice. Antibody titers were assessed serially by dot-blot analysis against specific peptides. Antibodies were affinity-purified by passage of immune serum over peptide columns prepared by covalent reaction of specific peptides with iodoacetyl-crosslinked agarose (Pierce). Antibodies were eluted at pH 2.5 and pH 11.5 followed by rapid titration at pH 8 and dialysis against PBS containing 0.02% sodium azide.Immunoblot Analysis. Organs from Sprague-Dawley rats were removed and homogenized in 200 mM sucrose/10 mM Tris-HCl, pH 7.4, containing leupeptin (1 ,tg/ml), pepstatin A (1 jig/ml), and antipain (4 ,Lg/ml). After homogenization in a Potter-Elvehjem apparatus and centrifugation at 3000 x g for 10 min, a high-speed pellet was prepared by centrifugation at 100,000 x g for 60 min. Membranes were dissolved in SDS Abbreviations: MIWC, mercurial-insensitive water channel; GLIP, glycerol intrinsic protein; CHIP, channel-forming integral protein.
Improperly folded membrane proteins are retained in the endoplasmic reticulum and then diverted to a degradative pathway by a network of molecular chaperones and intracellular proteases. Here we report that mutant insulin proreceptors (Pro 62 ) retained in the early secretory pathway undergo proteolytic cleavage at a tetrabasic concensus site for the subtilisin-like protease furin (SPC 1), generating two unstable proteolytic intermediates of 80͞120 kDa corresponding to ␣ (135 kDa) and  (90 kDa) subunits. These are degraded more rapidly than the uncleaved proreceptor protein.Site-directed mutagenesis of the normal RKRR processing site prevented cleavage. Use of inhibitors and furin-deficient cell lines confirmed that furin is responsible for proreceptor cleavage; furin overexpression increased the degradation of mutant but not wildtype receptors. Together, these results suggest that processing and degradation occur sequentially for mutant proreceptors.A central question in cell biology is how do eukaryotic cells distinguish and eliminate improperly folded proteins from the secretory pathway, a process referred to as biosynthetic quality control. Recent evidence indicates that degradation occurs after translocation from the endoplasmic reticulum (ER) to the cytosol and involves the 26S proteasome (1-11). However, the mechanism by which integral membrane proteins might be extruded from the ER remains unclear. Pharmacologic studies are beginning to support a role for proteases in biosynthetic quality control, including those with both trypsin and chymotrypsin-like activities, metalloproteinases, and the signal peptidase (12-17).Missense mutations in the insulin receptor (IR) that cause diabetes mellitus provide an example of a protein folding disorder in which cells recognize and destroy the abnormal protein (18)(19)(20). Studies of the biosynthesis of the wild-type IR have revealed that it initially is synthesized as a single chain proreceptor, which slowly folds and dimerizes in the ER and then is transported to the trans-Golgi where it is cleaved into two ␣ and two  subunits (21, 22). The mature subunits acquire sialic acid before transport to the cell surface (21-25). Misfolded receptors diverge from this pathway and are instead retained in the ER.We previously found that two proteins of 120 and 80 kDa accumulated in cells producing mutant receptors (26). Here we show that the 120-and 80-kDa proteins are immunoreactive with antireceptor antibodies, and they are generated by cleavage of the proreceptor. However, the 120-and 80-kDa proteins retain immature carbohydrates characteristic of localization in the ER or cis-Golgi. Radiosequencing revealed that cleavage of the proreceptor into 120͞80-kDa proteins occurs at a concensus site for the subtilisin-like protease, furin (RKRR2), an unexpected finding because furin was thought to act primarily in the transGolgi network (TGN). Together with recent work on the cleavage of the sterol regulatory element binding protein (27,28), the studies here suggest that pro-prot...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.