Specific Cellular Proteins Associate with Angiotensin-converting Enzyme and Regulate Its Intracellular Transport and Cleavage-Secretion

Abstract

Angiotensin-converting enzyme (ACE) is an extensively glycosylated type I ectoprotein anchored in the plasma membrane by a hydrophobic transmembrane domain. In tissue culture as well as in vivo, the extracellular domain of ACE is released into the culture medium by a regulated proteolytic cleavage. To identify the cellular proteins that regulate ACE processing and cleavagesecretion, ACE-bound proteins were purified by affinity chromatography and characterized by microsequencing and Western blotting. One protein was identified as ribophorin and another as immunoglobulin-binding protein (BiP), a chaperone. Metabolic labeling and immunoprecipitation of ACE confirmed its interaction with BiP. Overexpression of BiP inhibited ACE secretion, an effect accentuated by the expression of an enzymatically inactive mutant BiP. This inhibition was caused by the retention of ACE precursors by BiP in the endoplasmic reticulum, as revealed by immunoprecipitation and immunofluorescence experiments. However, treatment with a phorbol ester, phorbol 12-myristate 13-acetate, enhanced ACE secretion even from cells overexpressing BiP. Western blot analysis of ACE-associated proteins with antibodies to protein kinase C (PKC) revealed the presence of its specific isozymes. Treatment with phorbol 12-myristate 13-acetate caused marked reduction in ACE association of selective PKC species. Thus, our studies have identified PKC and BiP as two proteins that directly interact with ACE and modulate its cellsurface expression and cleavage-secretion.

Ribophorin I acts as a substrate-specific facilitator of N-glycosylation

“…Binding to ribophorin I can be observed both in vitro and in vivo, and the interaction does not depend upon the Nglycosylation of the precursor protein (Wilson et al, 2005). Other studies also identified a strong interaction between ribophorin I, and newly synthesised membrane proteins (Lilley and Ploegh, 2004;Santhamma and Sen, 2000), and we speculated that ribophorin I could function to improve the efficiency of N-glycosylation of selected substrates (Wilson et al, 2005). To test this hypothesis, we have developed a novel assay to study the consequences of small interfering RNA (siRNA)-mediated depletion of ribophorin I, STT3A and STT3B upon the N-glycosylation of several different membrane and secretory proteins.…”

Increased Shedding of Angiotensin-converting Enzyme by a Mutation Identified in the Stalk Region

“…Because both the basal and phorbol ester-induced cleavage secretion of [Leu 1199 ]ACE resembled what is observed for the testicular isoform of ACE, we investigated the hypothesis that the mutation could suppress an effect of the N-terminal domain on solubilization. This was achieved by introducing the mutation in an ACE expression vector lacking the N-domain of ACE (pACE-CF) (16 It was recently shown that, in the ACE89 cell line, testicular ACE can make a complex with protein kinase C subunits, and this complex dissociates in the presence of phorbol ester (28). A subsequent enhanced effect of ACE secretase was proposed to explain the phorbol ester-induced cleavage-secretion.…”

μ-Opioid Receptor Cell Surface Expression Is Regulated by Its Direct Interaction with Ribophorin I

“…BiP is responsible for maintaining the permeability barrier of the ER during protein translocation, directing protein folding and assembly, and targeting misfolded proteins for retrograde translocation so they can be degraded by the proteasomes (Hendershot, 2004;Alder et al, 2005). Hsp70 (BiP), together with RPNI, already has been found to interact with the angiotensin-converting enzyme receptor (Santhamma and Sen, 2000). It is noteworthy that we identified one of the proteins copurified with (His)6-MOR to be Bip.…”