Identification of the extracellular loop 2 as the point of interaction between the N terminus of the chemokine MIP-1alpha and its CCR1 receptor.
Macrophage inflammatory peptide-1alpha (MIP-1alpha)/CC-chemokine receptor ligand 3 is an 8-kDa peptide that induces chemotaxis of various lymphocytes to sites of inflammation through interaction with the G protein-coupled chemokine receptors CCR1 and CCR5. We recently described the preparation of a photoactivatable derivative of MIP-1alpha labeled with a benzophenone group at the extreme N-terminal end, which is a determinant for the agonist character of chemokines. Benzophenone-MIP-1alpha is a full agonist that specifically and covalently labels CCR1 and CCR5 receptors upon irradiation. In the present study, we use enzymatic and chemical cleavage methods on wild-type and mutated CCR1 receptors to show that the N terminus of the chemokine MIP-1alpha interacts in a specific manner with the second extracellular loop of the CCR1 receptor, within a segment comprising amino acids 178 to 194. This is the first report on the direct identification of a contact point between the N terminus of a chemokine and its membrane-bound receptor. The work shows that the part of chemokines that is endowed with agonist properties interacts with extracellular parts of the receptor rather than the transmembrane core of the protein.
Département Récepteurs et Protéines Membranaires, Centre National de la Recherche Scientifique UPR 9050 and IFR 85, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
Sannah
S
Zoffmann
André
A
Chollet
Jean-Luc
JL
Galzi
D000595:Amino Acid Sequence; D064753:Ammonium Hydroxide; D000818:Animals; D001665:Binding Sites; D001704:Biopolymers; D016466:CHO Cells; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018925:Chemokines / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D003488:Cyanogen Bromide / Q000737:chemistry; D006878:Hydroxides / Q000737:chemistry; D019402:Macrophage Inflammatory Proteins / Q000737:chemistry / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D054389:Receptors, CCR1; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D001704:Biopolymers; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018925:Chemokines; D006878:Hydroxides; D019402:Macrophage Inflammatory Proteins; D019920:Photoaffinity Labels; D054389:Receptors, CCR1; D019707:Receptors, Chemokine; D064753:Ammonium Hydroxide; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; C044499:submandibular proteinase A; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D003488:Cyanogen Bromide