EP24.15 interacts with the angiotensin II type I receptor and bradykinin B2 receptor.
The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 receptor (AT1) is known to interact with several classes of intracellular proteins that may modulate receptor function. Employing yeast two-hybrid screening of a human embryonic kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the AT1 receptor as a bait, we have isolated EP24.15 (EC 3.4.24.15, thimet oligopeptidase) as a potentially interacting protein. EP24.15 is widely distributed and is known to degrade bioactive peptides such as angiotensin I and II and bradykinin. In addition, EP24.15 was previously identified as a putative soluble angiotensin II binding protein. Two-hybrid screening also determined that EP24.15 can interact with the B2 bradykinin receptor. Transient expression of EP24.15 in a porcine kidney epithelial cell line stably expressing full length AT1 and full length B2 followed by affinity chromatography and co-immunoprecipitation confirmed EP24.15 association with both AT1 and B2 receptors. EP24.15 was also co-immunoprecipitated with AT1 and B2 in rat kidney brush border membranes (BBM) and basolateral membranes (BLM). Both AT1 and B2 undergo ligand-induced endocytosis. Analysis of endosomal fractions following immunoprecipitation with AT1 or B2 antibodies detected strong association of EP24.15 with the receptors in both light and heavy endosomal populations. Therefore, the present study indicates that EP24.15 associates with AT1 and B2 receptors both at the plasma membrane and after receptor internalization and suggests a possible mechanism for endosomal disposition of ligand that may facilitate receptor recycling.
Shivakumar
Bangalore R
BR
Department of Medicine, Vanderbilt University and Veterans Affairs Medical Center Nashville, TN 37232, USA.
Wang
Zoufei
Z
Hammond
Timothy G
TG
Harris
Raymond C
RC
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D011992:Endosomes / Q000201:enzymology; D015723:Gene Library; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007672:Kidney Cortex / Q000166:cytology / Q000201:enzymology; D018374:LLC-PK1 Cells; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D017434:Protein Structure, Tertiary; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D043782:Receptor, Bradykinin B2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013552:Swine; D020798:Two-Hybrid System Techniques
D044140:Receptor, Angiotensin, Type 1; D043782:Receptor, Bradykinin B2; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase