The type 1 angiotensin II receptor tail affects receptor targeting, internalization, and membrane fusion properties

Endocytosis modulates cell responses by removing and recycling receptors from the cell surface. Type I angiotensin II receptors (AT(1)R) are somewhat unique in that they are expressed at apical (AP) and basolateral (BL) membranes in proximal tubule cells and both receptor sites undergo endocytosis. We analyzed AT(1)R cytoplasmic (-COOH) tail deletion mutants to determine whether classic AT(1)R endocytosis motifs functioned similarly in polarized cells and simultaneously altered receptor properties. Serially truncating the AT(1)R tail had little effect on AP/BL AT(1)R distribution as determined by I-125-angiotensin II binding in LLCPKCl4 cells transfected with an AT(1)R transcript. AP AT 1 R expression required the proximal 12 amino acids in the AT(1)R-COOH tail. Deleting all but the proximal 12 aa of the AT(1)R-COOH tail (T316L mutant) decreased AP AT(1)R internalization at 20 min ( 17 +/- 6%; p < 0.05 versus full-length; n = 5) and inhibited AP AT(1)R-stimulated arachidonic acid release ( counts released per milligram of protein at 20 min: full-length, 18,762 +/- 4018; T316L, 2430 +/- 1711; n = 4; p < 0.02). Endosomal fusion assays were performed using peptide sequences of regions in the AT(1)R tail involved in endocytosis (YFLQLLKYIPP [LL] and LSTKMSTLSY [STL]). Peptide STL significantly inhibited endosomal fusion (22 +/- 10% of control; n = 5; p < 0.05 versus positive control). Peptide LL had no significant inhibitory effect. AT(1)R in polarized cells contain dominant endocytosis signals but these motifs do not correlate with AP or BL AT(1)R expression. Moreover, peptide sequences within the AT(1)R - COOH tail necessary for endocytosis also modulate endosomal fusion properties.