As the experimental procedure used could reflect agonist

As the experimental procedure used could reflect agonist-stimulation of both anterograde and retrograde APJ trafficking, as has been described for the δ-opioid peptide receptor (Zhang et al., 2006b, Zhang et al., 2006a), receptor internalization was more directly monitored by loading cell surface HA-mAPJ with anti-HA antibody before washing and incubation with agonist. We found a rapid increase in [Pyr1]apelin-13-induced HA-mAPJ internalization that is consistent with previous confocal microscopy studies showing rapid agonist-induced internalization of eGFP-APJ from the plasma membrane (Lee et al., 2010, El Messari et al., 2004). [Pyr1]apelin-13-induced-HA-mAPJ internalization was inhibited in the presence of sucrose, which prevents formation of clathrin-coated pits (Heuser and Anderson, 1989), and by the expression of DNM cDNAs of GRK2, dynamin and EPS15, known effectors of CME, but not by expression of βARRDNM. We have used these DNMs successfully to assess GRK2-, β-arrestin1-, dynamin- and EPS-dependent internalization of several GPCRs (e.g. oxytocin receptor (Smith et al., 2006); gonadotropin-releasing hormone receptor (Hislop et al., 2001, Hislop et al., 2005)). The GRK2DNM construct (K220A) reduces agonist-induced GPCR phosphorylation (Mundell et al., 1997); βARRDNM (319–418) competes with wild-type arrestin for clathrin and AP2 binding, and impairs receptor-binding ability (Krupnick et al., 1997); EPSDNM (EΔ95/295) lacks domains recognising EPS15 itself and is required for coated pit formation; and DYNDNM (K44A) inhibits dynamin-mediated scission of CCVs from the plasma membrane (Damke et al., 1994). Together these data suggest that [Pyr1]apelin-13 causes internalization of mAPJ via CCVs, and thereby reduces cell surface mAPJ levels. In accord with many other GPCRs, we suggest that GRK2-mediated phosphorylation targets the receptors to CCVs that are internalized from the cell surface in an EPS15- and dynamin-dependent manner. Interestingly, apelin-13-internalized APJ has been reported to dissociate from β-arrestin1 prior to receptor internalization (Lee et al., 2010), and we have shown that the [Pyr1]apelin-13 effect on inclusion count was not prevented by transfection with the βARRDNM cDNA. Although not tested, it is likely that both β−arrestin1- and β−arrestin2-mediated cetp inhibitors would be inhibited by transfection of the βARRDNM construct (J.L. Benovic, personal communication). Further work such as using siRNAs specifically against each arrestin will determine whether either is necessary for APJ-mediated internalization. Therefore the means by which these receptors are targeted to CCVs for internalization remains unclear. A similar dynamin-dependent, β-arrestin independent internalization has been reported in the 5-hydroxytryptamine 2A (5-HT2A) receptor (Bhatnagar et al., 2001). Desensitization of APJ has been shown not to occur in C-terminally truncated receptors that lack the majority of the serine and threonine residues that are liable to phosphorylation (Masri et al., 2006). Phosphorylation of GPCRs following ligand activation is the first step in receptor desensitization, occurring rapidly upon exposure to the agonist, and is conducted by second messenger kinases (e.g. protein kinase C (Benovic et al., 1985)) and a family of kinases termed GRKs (Benovic et al., 1986). Transfection of GRK2DNM or DYNDNM cDNA constructs into mAPJ-HEK293 cells pretreated with [Pyr1]apelin-13 for 2 h, both of which constructs had prevented HA-mAPJ internalization, did not affect the abrogation of subsequent responses to stimulation with [Pyr1]apelin-13. This data suggests that internalization is not a major factor in the desensitization of APJ-mediated ERK activation in these cells, and that desensitization of APJ is not dependent upon phosphorylation of APJ by GRK-2, but likely by kinases other than GRK-2. Focusing on recovery after agonist removal, pre-incubation of HA-mAPJ-HEK293 cells with [Pyr1]apelin-13 and subsequent recovery in agonist-free medium for varied periods revealed that cell surface APJ levels recovered to near control levels at 4–6 h, while APJ levels in the whole cell remained low following up to 6 h of recovery. Similarly, recovery in agonist-free medium rapidly reversed the number of inclusions seen after pre-incubation with [Pyr1]apelin-13 for 2 h. These data are generally consistent with agonist-induced internalization and reduction in cell surface HA-mAPJ occurring relatively rapidly during agonist exposure and recovering more slowly after agonist removal. They also reveal two unexpected features. First, the proportion of HA-mAPJ at the cell surface 2 h after removal of the [Pyr1]apelin-13 pre-treatment is significantly higher than in control cells prior to [Pyr1]apelin-13 pre-treatment (∼98% versus ∼76%), suggesting that all available APJ are specifically compartmentalized at the cell surface upon agonist removal. Second, the rate of loss of inclusions during the recovery period (half-time ∼15 min) was much higher than the rate of recovery of cell surface HA-mAPJ (half-time ∼2 h). This may suggest that inclusions are lost as the anti-HA shifts from early endosomes to a sorting compartment. If so, their loss would be expected to precede recovery of cell surface receptor expression. In following recovery from desensitization we transfected GRK2DNM, DYNDNM and βARRDNM cDNAs into mAPJ-HEK293 cells and found delayed resensitization of [Pyr1]apelin-13-induced ERK1/2 responses after transfection with GRK2DNM and DYNDNM cDNAs, but not with the βARRDNM cDNA. This indicates that GRK2- and dynamin-dependent receptor internalization may have a role to play in the resensitization of [Pyr1]apelin-13-induced ERK phosphorylation, however β−arrestin-dependent internalization is not required for this process. Thus regulation of receptor number may determine responsiveness of mAPJ to repeated [Pyr1]apelin-13 stimulation in HEK293 cells.