Apical targeting of the P2Y(4) receptor is directed by hydrophobic and basic residues in the cytoplasmic tail.
The P2Y(4) receptor is selectively targeted to the apical membrane in polarized epithelial cell lines and has been shown to play a key role in intestinal chloride secretion. In this study, we delimit a 23 amino acid sequence within the P2Y(4) receptor C-tail that directs its apical targeting. Using a mutagenesis approach, we found that four hydrophobic residues near the COOH-terminal end of the signal are necessary for apical sorting, whereas two basic residues near the NH(2)-terminal end of the signal are involved to a lesser extent. Interestingly, mutation of the key hydrophobic residues results in a basolateral enrichment of the receptor construct, suggesting that the apical targeting sequence may prevent insertion or disrupt stability of the receptor at the basolateral membrane. The signal is not sequence specific, as an inversion of the 23 amino acid sequence does not disrupt apical targeting. We also show that the apical targeting sequence is an autonomous signal and is capable of redistributing the normally basolateral P2Y(12) receptor, suggesting that the apical signal is dominant over the basolateral signal in the main body of the P2Y(12) receptor. The targeting sequence is unique to the P2Y(4) receptor, and sequence alignments of the COOH-terminal tail of mammalian orthologs reveal that the hydrophobic residues in the targeting signal are highly conserved. These data define the novel apical sorting signal of the P2Y(4) receptor, which may represent a common mechanism for trafficking of epithelial transmembrane proteins.
Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
D Ross
DR
DuBose
Samuel C
SC
Wolff
Ai-Dong
AD
Qi
Izabela
I
Naruszewicz
Robert A
RA
Nicholas
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D016764:Cell Polarity / Q000502:physiology*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D008565:Membrane Proteins; D018048:Receptors, Purinergic P2; C104749:purinoceptor P2Y4