Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands David A. Zidara, Jonathan D. Violina, Erin J. Whalena, and Robert J. Lefkowitza,b,c,1 Departments of aMedicine, bBiochemistry, and cHoward Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710 Contributed by Robert J. Lefkowitz, April 24, 2009 (sent for review February 13, 2009) CCL19 and CCL21 are endogenous agonists for the seven-trans- to be uniquely required for ␤-arrestin-mediated MAP kinase membrane receptor CCR7. They are equally active in promoting G signaling (16, 17). protein stimulation and chemotaxis. Yet, we find that they result The chemokine receptor CCR7 is particularly important for in striking differences in activation of the G protein-coupled immune responses requiring the coordinated interaction of receptor kinase (GRK)/ß-arrestin system. CCL19 leads to robust various cell types within lymphoid tissues (18, 19). It is unique CCR7 phosphorylation and ␤-arrestin2 recruitment catalyzed by for its high expression on mature, naïve T lymphocytes and is both GRK3 and GRK6 whereas CCL21 activates GRK6 alone. This vital for homeostatic recirculation of these cells through sec- differential GRK activation leads to distinct functional conse- ondary lymphoid tissues. CCR7 has two endogenous ligands, quences. Although each ligand leads to ␤-arrestin2 recruitment, CCL19 and CCL21, which have evolved from a series of complex only CCL19 leads to redistribution of ␤-arrestin2-GFP into endocytic gene duplications and retain 32% amino acid sequence homol- vesicles and classical receptor desensitization. In contrast, these ago- ogy (20–22). They are predicted to be structurally similar except ␤ nists are both capable of signaling through GRK6 and -arrestin2 to that CCL21 has a conserved motif consisting of an additional 37 ERK kinases. Thus, this mechanism for ‘‘ligand bias’’ whereby amino acids at its C terminus (23). CCL19 and CCL21 have been endogenous agonists activate different GRK isoforms leads to shown to have similar binding affinities (Kd Ϸ100 pM), equal ␤ BIOCHEMISTRY functionally distinct pools of -arrestin. efficacy for G protein activation, calcium flux, and chemotactic responses (20–22, 24–26). Yet, some signaling characteristics ͉ ͉ ͉ ͉ CCR7 CCL19 CCL21 chemokine arrestin appear to be unique to CCL19-activated CCR7. For instance, CCL19 but not CCL21 is reported to lead to receptor desensi- raditional agonists of seven-transmembrane receptors tization (20), internalization (27, 28), receptor degradation (29), T(7TMRs) lead to the simultaneous activation of G protein- and rapid dendrite extension (30). Recently, Kohout et al. (31) ␤ and -arrestin-dependent signaling pathways through a series of have reported that CCL19 but not CCL21 also leads to receptor broadly conserved biochemical steps (1). Receptor activation phosphorylation and ␤-arrestin recruitment. Here, we examine promotes the dissociation of heterotrimeric G proteins into free ␥ the role of individual GRKs in orchestrating the differential G␣ and G␤ subunits and subsequent second messenger signal- signaling of CCR7 with particular emphasis on ␤-arrestin- ing. Agonist-activated 7TMRs undergo phosphorylation by G mediated functions. protein-coupled receptor kinases (GRKs), and this promotes the binding of ␤-arrestins (2). ␤-Arrestins desensitize 7TMR- Results mediated G protein signaling at several mechanistic levels G Protein Signaling. CCL19 and CCL21 have been shown to have including direct steric hindrance of the receptor by ␤-arrestin i/o equal binding affinities for CCR7 and equivalent efficacy and attachment (3), recruitment of 2nd messenger-degrading en- potency for the activation of the G subfamily of G proteins (21, zymes (4, 5), and by acting as a scaffold for proteins that facilitate i/o 24, 25). To confirm this in live cells, we designed an assay to study receptor internalization (6). G -mediated inhibition of adenylate cyclase by using the cAMP ␤-Arrestin binding also initiates additional signaling programs i/o through coordinated recruitment of signaling intermediates to biosensor ICUE2 (32, 33). Human embryonic kidney (HEK) ␤ cells stably coexpressing CCR7 and ICUE2 were treated with activated receptors. For instance, -arrestin-dependent signaling ␮ to MAP kinase has been extensively characterized and is de- forskolin (50 M) in the absence or presence of saturating monstrable for many 7TMRs (7, 8). Proteomic approaches concentrations of CCL19 (100 nM) or CCL21 (100 nM). Fors- predict an ‘‘interactome’’ of hundreds of additional binding kolin stimulation results in a large decrease in ICUE2 FRET, partners (9). indicating an increase in intracellular cAMP concentration (Fig. Recently, ‘‘␤-arrestin-biased’’ synthetic ligands have been 1A). Coincubation of forskolin with either CCR7 ligand led to a blunted response indicative of simultaneous G signaling. There described for the angiotensin 1A receptor (10), ␤2-adrenergic i/o receptor (␤2AR) (11, 12), and parathyroid hormone receptor was no difference between CCL19 and CCL21 for the inhibition (13). These ligands each cause receptor phosphorylation and of forskolin responsiveness, indicating that these ligands have ␤-arrestin-mediated effects in the absence of G protein activa- comparable efficacy for Gi/o signaling. tion. The mechanism of ligand bias is poorly understood al- though biophysical studies suggest that agonists and receptors Author contributions: D.A.Z., J.D.V., E.J.W., and R.J.L. designed research; D.A.Z. performed oscillate among multiple conformations, some of which may be research; D.A.Z., J.D.V., E.J.W., and R.J.L. analyzed data; and D.A.Z., J.D.V., E.J.W., and R.J.L. capable of differential signaling (14, 15). wrote the paper. ␤ Given the complexity of -arrestin function, the traditional The authors declare no conflict of interest. concept of GRKs as redundant kinases is likely overly simplistic. Freely available online through the PNAS open access option. GRKs 2, 3, 5, and 6 are ubiquitously expressed. The extent to 1To whom correspondence should be addressed at: Box 3821, Duke University Medical which they provide redundancy versus functional specialization Center, Durham, NC 27710. E-mail: [email protected]. is largely unknown, but evidence for the latter has recently This article contains supporting information online at www.pnas.org/cgi/content/full/ emerged in some receptor systems. For instance, GRK5/6 appear 0904361106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904361106 PNAS ͉ June 16, 2009 ͉ vol. 106 ͉ no. 24 ͉ 9649–9654 Downloaded by guest on September 28, 2021 ABC 9 -10 -10 CCL19 7 -8 -8 NS CCL19CCL21 FSK 5 -6 -6 FSK IP: CCR7- CCL21 -4 -4 FLAG 3 %FRET -2 FSK+CCL19 -2 FSK+CCL21 1 CXCL12 ICUE2 %FRET ICUE2 %FRET 0 0 2.5 5.0 7.5 10.0 2.5 5.0 7.5 10.0 2.5 5.0 7.5 10.0 -1 Minutes Minutes Minutes 90 100 80 60 *** 70 75 50 60 *** 40 50 50 40 30 *** CCR7 30 ** -arrestin 20 β (%CCL19) 20 25 Recruitment (AUC, %FRET) (AUC, (AUC, %FRET) 10 10 Phosphorylation cAMP Accumulation cAMP 0 0 0 __ FSK + + + _ _ CXCL12 + _ _ CCL19 + _ _ CCL19 + __ CCL19 + _ _ CCL21 + _ _ CCL21 + CCL21 + Fig. 1. CCL19 and CCL21 lead to equivalent G protein responses but differential CCR7 phosphorylation and ß-arrestin2 recruitment. (A) HEK-293 cells stably expressing CCR7 and the cAMP biosensor ICUE2 were stimulated with 50 ␮M forskolin in the presence or absence of saturating concentrations of CCL19 (100 nM) or CCL21 (100 nM). cAMP accumulation was measured as the change in ICUE2 FRET ratio. (Upper) Change in FRET (mean Ϯ SE, n ϭ 4). (Lower) Integrated response 32 after 10 min. (B) HEK-293 cells stably expressing CCR7-FLAG were labeled with Pi and stimulated at 37 °C for 10 min with or without 100 nM CCL19 or 100 nM 32 CCL21 as indicated. (Upper) Representative image showing Pi incorporation. (Lower) Bar graph that represents the mean Ϯ SE from six independent experiments. (C) Live HEK-293 cells stably expressing CCR7-CFP and ß-arrestin2-YFP were stimulated with 100 nM CXCL12, 100 nM CCL21, or 100 nM CCL19, and the recruitment of ß-arrestin2 was measured by FRET. Data represent the mean Ϯ SE from six experiments. Statistical significance was determined by paired two-tailed t tests. **, P Ͻ 0.01; ***, P Ͻ 0.001. CCR7 Phosphorylation and ␤-Arrestin2 Recruitment. The ability to nor class B, suggesting that this interaction is even more transient undergo agonist-induced phosphorylation is a hallmark of than that typically seen for class A receptors. 7TMRs and an important, proximate indicator of GRK function. ␤-Arrestin recruitment classically leads to agonist-induced To compare CCL19 and CCL21 for bulk receptor phosphory- receptor desensitization. To compare the CCR7 ligands for their 32 lation, we analyzed the incorporation of Pi into CCR7-FLAG ability to induce desensitization, HEK cells stably expressing after 10 min of stimulation. CCL19 (100 nM) led to robust CCR7 CCR7 and ICUE2 were preincubated with CCL19 (100 nM) or phosphorylation (Fig. 1B), whereas CCL21 (100 nM) mediated CCL21 (100 nM) for 60 min. Cells were then washed, and Gi/o a much smaller but consistent level of CCR7 phosphorylation activity was assessed by restimulation with CCL21 (100 nM) and Ϯ (21.6 6.1% of that induced by CCL19). forskolin (50 ␮M). We found that preincubation with CCL19 ␤ Next, we sought to compare -arrestin2 recruitment with leads to complete desensitization of CCR7 toward subsequent G CCR7 in response to CCL19 and CCL21. Live HEK cells stably protein activation (Fig. 2B). In contrast, desensitization was not ␤ expressing CCR7-CFP and -arrestin2-YFP were stimulated observed after preincubation with CCL21. with 100 nM CCL19 or 100 nM CCL21, and the time course of ␤ -arrestin recruitment to the receptor was analyzed by FRET ␤-Arrestin2-Mediated MAPK Signaling. In addition to desensitiza- (Fig. 1C). CCL19 promoted a robust interaction between the tion, ␤-arrestins also function as scaffolds to facilitate signaling receptor and ␤-arrestin2 whereas the effect of CCL21 was weak pathways such as ERK activation.