Received for publication September 20, 2004.
Revised February 2, 2005.
Accepted for publication February 2, 2005.

Subtype-Specific Sorting of The ET_A Endothelin Receptor by A Novel Endocytic Recycling Signal for G Protein-Coupled Receptors

Abstract

We have previously reported that endocytic sorting of ET_A endothelin receptors to the recycling pathway is dependent on a signal residing in the cytoplasmic carboxyl-terminal region. The aim of the present work was to characterize the carboxyl-terminal recycling motif of the ET_A receptor. Assay of truncation mutants of the ET_A receptor with increasing deletions of the carboxyl-terminal tail revealed that amino acids 390 - 406 contained information critical for the ability of the receptor to recycle. This peptide sequence displayed significant sequence similarity to several protein segments confirmed by X-ray crystallography to adopt antiparallel -strand structures (-finger). One of these segments was the -finger motif of nNOS (neuronal nitric oxide synthase) reported to function as an internal PDZ (PSD-95/Dlg/ZO-1) domain-binding ligand (PDZ ligand). Based on these findings, the three-dimensional structure of the recycling motif of ET_A receptor was predicted to attain a -finger conformation acting as an internal PDZ ligand. Site-directed mutagenesis at residues that would be crucial to the structural integrity of the putative -finger conformation or PDZ ligand function prevented recycling of the ET_A receptor. Analysis of more than 300 G protein-coupled receptors (GPCRs) identified 35 different human GPCRs with carboxyl-terminal sequence patterns that fulfilled the structural criteria of an internal PDZ ligand. Among these are several receptors reported to follow a recycling pathway. In conclusion, recycling of ET_A receptor is mediated by a motif with the structural characteristics of an internal PDZ ligand. This structural motif may represent a more general principle of endocytic sorting of GPCRs.

Key words: Endothelin, Sequestration/Internalization, Recycling, Structure determinations, Structure-activity relationships and modeling

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