Endothelin-converting enzyme-1 regulates endosomal sorting of calcitonin receptor-like receptor and β-arrestins

Abstract

Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a–d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), β-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and β-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and β-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B2 receptor, which transiently interacts with β-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/β-arrestin complex, freeing internalized receptors from β-arrestins and promoting recycling and resensitization.

Endosomal Endothelin-converting Enzyme-1

“…PD-069185 (19) was a gift (Pfizer). Other reagents were from Sigma or have been described (12,13,20).…”

“…Measurement of [Ca 2ϩ ] i -[Ca 2ϩ ] i was measured in populations of cells using Fura2-AM (Invitrogen) as described (12,20). To assess resensitization, cells were incubated with vehicle or SP (10 nM, 0 or 10 min, 37°C), washed, and recovered in SP-free medium for 2 h. Cells were challenged with SP (10 nM), and [Ca 2ϩ ] i was measured.…”

“…Transfected Cells and Cell Lines-Generation and maintenance of human embryonic kidney 293 (HEK) FLP cells (Invitrogen) and HEKFLP stably expressing rat NK 1 R have been described (12,13,20). In some experiments cells were transiently transfected with rat NK 1 R, rat FLAG-NK 1 R⌬325-407, rat ␤-arrestin1 V53D, rat ␤-arrestin1 319 -418 , human ␤-arrestin2-GFP, human ECE-1c-GFP, or empty vector (control) using Lipofectamine2000 (Invitrogen).…”

“…Membranes were incubated with antibodies to pERK2 (E-4; 1:1,000), ERK2 (C-14; 1:5,000), c-Fos (9F6, 1:2,000), ECE-1 (1:1,000), EEA1 (1:10,000), Rab5a (1:2,000), LAMP1 (1:2,000), Na ϩ -K ϩ -ATPase (1:5,000), histone H3 (1:20,000), NK 1 R (1:10,000), ␤-arrestin2 (1:5,000), Src (1:1,000), Nur77 (1:200), phosphothreonine (1:1,000), or ␤-actin (A-5441, 1:10,000) overnight at 4°C. Membranes were treated with secondary or tertiary antibodies and analyzed with an Odyssey Infrared Imaging System (Li-COR Biosciences) (12,13,20).…”

“…We hypothesized that mechanisms that regulate GPCRs at the plasma membrane also control receptor signaling and trafficking at the endosomal membrane. We recently reported that the metalloendopeptidase endothelin-converting enzyme-1 (ECE-1) is present at the endosomal membrane and that ECE-1 degrades internalized neuropeptides within acidified early endosomes (12)(13)(14). This degradation disrupts the peptide-GPCR-␤-arrestin complex to promote ␤-arrestin translocation back to the cytosol and receptor recycling to the cell-surface, which mediates resensitization.…”