Determinants of High-Affinity Binding and Receptor Activation in the N-Terminus of CCL-19 (MIP-3β)

Abstract

CC chemokine receptor 7 (CCR-7) is expressed on mature dendritic cells and T-cells. Its ligands, CCL-19 (MIP-3beta) and CCL-21 (SLC), play an important role in the migration of these cells to secondary lymphoid organs where they are predominantly expressed. For most chemokines, the N-terminal domain preceding the first two conserved cysteines is involved in stabilizing the active conformation of its cognate receptors. We have chemically synthesized N-terminal analogues of CCL-19 with the aid of a native chemical ligation method to investigate structure function requirements of this ligand domain by performing ligand binding, GTP-gammaS binding, and chemotaxis assays. Successive truncations of the N-terminus of CCL-19 reduced the affinity of the receptor for the ligand in a size-dependent manner. Furthermore, Ala substitutions of Asn(3), Asp(4), and Asp(7) show that the side chains of these residues are important for high-affinity binding of CCL-19 to CCR-7. The effects observed were mirrored in both GTP-gammaS binding and chemotaxis assays, highlighting the functional importance of this ligand domain. We also describe two partial agonists of CCR-7 ([Nle(72)]CCL-19(6-77) and Ac-[Nle(72)]CCL-19(7-77)), and identify the first analogue of CCL-19 (Ac-[Nle(72)]CCL-19(8-77)) that acts as a functional antagonist in vitro (K(B) approximately 350 nM for GTP-gammaS binding assays). As mutations of both Glu(6) and Asp(7) to Ala did not dissociate effects on ligand binding from receptor activation, it is likely that the backbone of these two residues is crucial for agonist activity.

Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands

“…This motif could account for the differential signaling described here by a variety of mechanisms, including tethering CCL21 to extracellular glycosaminoglycans or by differentially inducing ligand oligomerization. Alternatively, because the amino acids of the N terminus of CCL19 have been shown to regulate receptor activation (36)(37)(38), subtle differences in the primary structure in this region may account for differential signaling. Thus, additional studies using chimeric ligands could test the relative importance of these motifs for CCR7 ligand bias.…”

Identification of CC Chemokine Receptor 7 Residues Important for Receptor Activation

“…It is likely that the high affinity interactions between the N-loop of the ligand, and the N terminus and/or extracellular loop domains of the receptor position the ligand in an appropriate orientation, so that crucial contacts can be made with residues in the helix bundle of the receptors, which stabilize the receptor in the active conformation (17)(18)(19). We have previously shown in CCR-7 that the N-terminal domain of CCL-19 is involved in both high affinity ligand binding and receptor activation (20). With the aid of sequence alignments we identified five residues near the extracellular surface of CCR-7 that appeared likely to be involved in stabilizing the active conformation.…”

“…The above mechanisms, however, can vary between different chemokines, as e.g. CCL-19 (MIP-3␤) (20) and CXCL-12 (SDF-1) (19) contain residues important for high affinity binding in the N terminus, while CCL-11 (Eotaxin-1) contains residues important for receptor activation in the Nloop (21).…”

“…We previously reported that the N terminus of CCL-19 plays a central role in high affinity binding and receptor activation (20) and while CCL-19 and CCL-21 share little sequence homology, a conserved Asp is found immediately preceding the CC motif in both chemokines, which was shown to be crucial for receptor activation (20 (38,39). However, when Asp 7 in CCL-19 was mutated to Ala, binding affinity to the wild-type receptor was reduced 70-fold and potency for G protein activation was reduced 250-fold (20), which is inconsistent with the present result.…”

“…We previously reported that the N terminus of CCL-19 plays a central role in high affinity binding and receptor activation (20) and while CCL-19 and CCL-21 share little sequence homology, a conserved Asp is found immediately preceding the CC motif in both chemokines, which was shown to be crucial for receptor activation (20 (38,39). However, when Asp 7 in CCL-19 was mutated to Ala, binding affinity to the wild-type receptor was reduced 70-fold and potency for G protein activation was reduced 250-fold (20), which is inconsistent with the present result. Truncation of the N terminus of CCL-19 showed that it is likely that there is an interaction between the backbone of Glu 6 or Asp 7 of CCL-19 and CCR-7, required for activation (20), and it is possible that Lys 3.26 (130) interacts with the backbone of one of these residues.…”