Determinants of high-affinity binding and receptor activation in the N-terminus of CCL-19 (MIP-3 beta).
Biochemistry
; 43(12): 3670-8, 2004 Mar 30.
Article
en En
| MEDLINE
| ID: mdl-15035637
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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Fragmentos de Péptidos
/
Receptores de Quimiocina
/
Quimiocinas CC
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Biochemistry
Año:
2004
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos