Replacement of the Transmembrane Anchor in Angiotensin I-converting Enzyme (ACE) with a Glycosylphosphatidylinositol Tail Affects Activation of the B2 Bradykinin Receptor by ACE Inhibitors

Branislav Marcic,Peter A Deddish,Randal A Skidgel,Ervin G Erdös,Richard D Minshall,Fulong Tan

doi:10.1074/jbc.m909490199

Abstract

To investigate further the relationship of angiotensin I-converting enzyme (ACE) inhibitors to activation of the B(2) bradykinin (BK) receptor, we transfected Chinese hamster ovary cells to stably express the human receptor and either wild-type ACE (WT-ACE), an ACE construct with most of the cytosolic portion deleted (Cyt-del-ACE), or ACE with a glycosylphosphatidylinositol (GPI) anchor replacing the transmembrane and cytosolic domains (GPI-ACE). BK or its ACE-resistant analogue were the agonists. All activities (arachidonic acid release and calcium mobilization) were blocked by the B(2) antagonist HOE 140. B(2) was desensitized by repeated administration of BK but resensitized to agonist by ACE inhibitors in the cells expressing both B(2) and either WT-ACE or Cyt-del-ACE. In GPI-ACE expressing cells, the B(2) receptor was still activated by the agonists, but ACE inhibitors did not resensitize. Pretreatment with filipin returned the sensitivity to inhibitors. In immunocytochemistry, GPI-ACE showed patchy, uneven distribution on the plasma membrane that was restored by filipin. Thus, ACE inhibitors were inactive as long as GPI-ACE was sequestered in cholesterol-rich membrane domains. WT-ACE and B(2) receptor in Chinese hamster ovary cells co-immunoprecipitated with antibody to receptor, suggesting an interaction on the cell membrane. ACE inhibitors augment BK effects on receptors indirectly only when enzyme and receptor molecules are sterically close, possibly forming a heterodimer.

Highlights

Renin was discovered over a century ago [1], and kallikrein was discovered about 25 years later [2]
Investigations using cultured cells transfected with cDNA of the human B2 receptor provided evidence that angiotensin I-converting enzyme (ACE) inhibitors do not act on the B2 receptor directly but enhance BK effects and resensitize the receptor to BK only if ACE is present on the plasma membranes of cells [18, 19]
These experiments indicate that the cytosolic and transmembrane domains are not required for activation of the B2 receptor by ACE inhibitors, but ACE has to be in the immediate vicinity of the receptor for the activation to occur, possibly due to heterodimer formation

Summary

EXPERIMENTAL PROCEDURES

Materials—BK, benzoyl-Gly (hippuryl)-His-Leu (Hip-His-Leu), filipin, tissue culture medium, buffers and reagents were from Sigma. [Phe8(CH2NH)Arg9]bradykinin, an ACE-resistant BK analogue, was obtained from Novabiochem (San Diego, CA). To establish cell lines with stable expression, CHO cells were transfected with human mutant or wild-type ACE cDNA in a pcDNA3 expression vector (carrying the neomycin resistance gene) using the Superfect method with serum-free Ham’s F-12 medium without antibiotic, as described [19]. Different clones were harvested and propagated using cloning rings [19] Cells expressing both human B2 receptor and wild-type ACE were designated CHO/AB cells. Filipin Treatment—CHO cells expressing GPI-ACE and B2 receptor or B2 receptor alone were treated in monolayers with Ham’s F-12 medium (ϩ10% fetal bovine serum) containing 10 nM filipin for 30 min at 37 °C. Immunocytochemistry—Approximately 1 ϫ 106 CHO cells expressing B2 receptor and GPI-ACE were grown to confluence on coverslips and incubated for 2 h in phenol red-free and serum-free medium. Values of p Ͻ 0.05 were considered statistically significant

RESULTS

Activity Supernatant Pellet

DISCUSSION