Charged residues in the C-terminus of the P2Y1 receptor constitute a basolateral-sorting signal

Abstract

SummaryThe P2Y 1 receptor is localized to the basolateral membrane of polarized Madin-Darby canine kidney (MDCK) cells. In the present study, we identified a 25-residue region within the C-terminal tail (C-tail) of the P2Y 1 receptor that directs basolateral sorting. Deletion of this sorting signal caused redirection of the receptor to the apical membrane, indicating that the region from the N-terminus to transmembrane domain 7 (TM7) contains an apical-sorting signal that is overridden by a dominant basolateral signal in the C-tail. Location of the signal relative to TM7 is crucial, because increasing its distance from the end of TM7 resulted in loss of basolateral sorting. The basolateral-sorting signal does not use any previously established basolateral-sorting motifs, i.e. tyrosine-containing or dihydrophobic motifs, for function, and it is functional even when inverted or when its amino acids are scrambled, indicating that the signal is sequence independent. Mutagenesis of different classes of amino acids within the signal identified charged residues (five basic and four acidic amino acids in 25 residues) as crucial determinants for sorting function, with amidated amino acids having a lesser role. Mutational analyses revealed that whereas charge balance (+1 overall) of the signal is unimportant, the total number of charged residues (nine), either positive or negative, is crucial for basolateral targeting. These data define a new class of targeting signal that relies on total charge and might provide a common mechanism for polarized trafficking of epithelial proteins.

The role of aspartic acid residues 405 and 416 of the kidney isotype of sodium-bicarbonate cotransporter 1 in its targeting to the plasma membrane

“…These studies point out the important role of charged amino acid residues in the NH 2 terminus of NBCe1 in its accurate targeting to the plasma membrane. The role of charged amino acid moieties in the targeting of other proteins such as P2Y1 receptor to the membrane has been documented (40). The results presented in the present study and those describing the role of charged amino acids in the targeting of other membrane bound proteins suggest that such amino acid residues are important determi- (22,40).…”

“…Charged amino acid residues have been shown to play a role in the targeting of membrane bound proteins (40). To determine the role of the negative charge of aspartic acid residues 405 and 416 in the targeting of NBCe1A to the basolateral membrane, we examined the effect of their replacement with glutamic acid.…”

The iodide transport defect-causing mutation R124H: a δ-amino group at position 124 is critical for maturation and trafficking of the Na+/I− symporter (NIS)

“…R124 is the only charged residue in IL-2 that is essential for NIS targeting to the cell surface Intracellularly facing charged residues have been shown to be important in trafficking of membrane proteins (Kasahara et al, 2004;Li et al, 2012;Wolff et al, 2010). IL-2 contains three basic (R111, R124, R127) and two acid residues (E119, E122).…”

Identification of an intracellular microdomain of the P2X7 receptor that is crucial in basolateral membrane targeting in epithelial cells

“…3C). These results suggest that P570-Q587 and particularly P582-Q587 is critical in basolateral targeting of the P2X 7 R. Interestingly, a recent study has reported basolateral targeting of the ATP-sensitive metabotropic P2Y1 receptor that is also independent of l1B but critically requires a stretch of 25 residues in the distal intracellular C-terminus [14]. The findings from previous and present studies suggest that the receptors for extracellular ATP are directed to the basolateral membranes via motifs that are distinct from those previously identified [7].…”

“…Clearly, our results are consistent with these studies. The C-terminal part of this microdomain (P582-Y595) possesses a canonical basolateral targeting motif YxxU [7,14] (Y588-S589-G590-F591) (Fig. 3A).…”