By interacting with the C-terminal Phe of apelin, Phe255 and Trp259 in helix VI of the apelin receptor are critical for internalization.
Apelin is the endogenous ligand of the orphan seven-transmembrane domain (TM) G protein-coupled receptor APJ. Apelin is involved in the regulation of body fluid homeostasis and cardiovascular functions. We previously showed the importance of the C-terminal Phe of apelin 17 (K17F) in the hypotensive activity of this peptide. Here, we show either by deleting the Phe residue (K16P) or by substituting it by an Ala (K17A), that it plays a crucial role in apelin receptor internalization but not in apelin binding or in Gα(i)-protein coupling. Then we built a homology three-dimensional model of the human apelin receptor using the cholecystokinin receptor-1 model as a template, and we subsequently docked K17F into the binding site. We visualized a hydrophobic cavity at the bottom of the binding pocket in which the C-terminal Phe of K17F was embedded by Trp(152) in TMIV and Trp(259) and Phe(255) in TMVI. Using molecular modeling and site-directed mutagenesis studies, we further showed that Phe(255) and Trp(259) are key residues in triggering receptor internalization without playing a role in apelin binding or in Gα(i)-protein coupling. These findings bring new insights into apelin receptor activation and show that Phe(255) and Trp(259), by interacting with the C-terminal Phe of the pyroglutamyl form of apelin 13 (pE13F) or K17F, are crucial for apelin receptor internalization.
Iturrioz
Xavier
X
INSERM, U691, Collège de France, Université Pierre et Marie-Curie Paris 6, Paris FR-75005, France.
Gerbier
Romain
R
Leroux
Vincent
V
Alvear-Perez
Rodrigo
R
Maigret
Bernard
B
Llorens-Cortes
Catherine
C
D000595:Amino Acid Sequence; D000818:Animals; D000073861:Apelin; D000075244:Apelin Receptors; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism*; D011487:Protein Conformation; D011869:Radioligand Assay; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism*
C115436:APLN protein, human; C104575:APLNR protein, human; D000073861:Apelin; D000075244:Apelin Receptors; C491284:Aplnr protein, rat; D036341:Intercellular Signaling Peptides and Proteins; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D010649:Phenylalanine; D014364:Tryptophan; D000242:Cyclic AMP