By Interacting with the C-terminal Phe of Apelin, Phe255 and Trp259 in Helix VI of the Apelin Receptor Are Critical for Internalization

Abstract

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 G␣ 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-

Biased Signaling Favoring Gi over β-Arrestin Promoted by an Apelin Fragment Lacking the C-terminal Phenylalanine

“…We then investigated the ability of K17F and K16P to activate p42/44 MAPK (ERK) in CHO cells stably expressing the rat ApelinR in the absence or presence of pertussis toxin (PTX), a G i protein inhibitor, or in the absence or presence of a mutant of ␤-arrestin (␤-arrestin-2-K296A) that prevents the scaffolding of ERK1/2. Finally, we confirmed the involvement of ␤-arrestin-dependent ERK signaling pathway by using a mutated ApelinR previously shown to be unable to internalize upon K17F stimulation (28).…”

“…In contrast, the ability of K17A and K16P to induce ApelinR internalization is drastically reduced when compared with K17F, suggesting an important role for the C-terminal Phe of apelin in agonist-promoted receptor internalization (14,28).…”

“…The wild-type and mutated (F255A) rat ApelinRs tagged at their C-terminal part with YFP were constructed by subcloning the open reading frame of the wild-type or F255A ApelinR from previous constructs encoding the wild-type or F255A rat ApelinR tagged at its C-terminal part with EGFP (ApelinR-EGFP) (4,28) to pYFP-N1 (Clontech) using HindIII-BamHI restriction sites. C terminus HA-tagged wild-type and F255A-ApelinR were made by PCR using as template wild-type and F255/A-ApelinR-EGFP constructs and 5Ј-CATTGACGCAA-ATGGGCGGTAGGCGTG-3Ј and 5Ј-GGATCCTAAGCGT-AATCGGGCACATCGTAAGGGTAAGCGTAATCGGGC-ACATCGTAAGGGTAGTCCACAAGGGTTTCTTGGCTA-TAG-3Ј as forward primer and reverse primer, respectively.…”

“…We identified an hydrophobic cavity at the bottom of the binding pocket in which the C-terminal Phe of pE13F or K17F is embedded by Trp-152 in transmembrane IV and Trp-259 and Phe-255 in transmembrane VI. Site-directed mutagenesis revealed that Phe-255 and Trp-259 are required to trigger ApelinR internalization without playing a role in apelin binding nor in G i protein coupling (28).…”

“…We previously showed that the deletion or Ala substitution of the C-terminal Phe in K17F does not modify its ability to bind ApelinR or to inhibit forskolin-induced cAMP production (14,28), indicating that the C-terminal Phe of apelin does not play a major role in apelin binding to the receptor or its capacity to activate G i . In contrast, the ability of K17A and K16P to induce ApelinR internalization is drastically reduced when compared with K17F, suggesting an important role for the C-terminal Phe of apelin in agonist-promoted receptor internalization (14,28).…”