Identification of amino acids involved in the binding of hMIP-1α to CC-CKR1, a MIP-1α receptor found on neutrophils

Human macrophage inflammatory protein-1 alpha (hMIP-1 alpha) and human macrophage inflammatory protein-1 beta (hMIP-1 beta) are chemokines involved in a diverse range of immunological effects. Both hMIP-1 alpha and hMIP-1 beta are involved in the activation of monocytes and THP-1 cells probably through a common receptor(s). However, only hMIP-1 alpha can bind to neutrophils with high affinity, presumably through CC-CKR1 (CKR1). Since the structure of these two proteins is highly conserved, non-conserved amino acids must define the disparate binding patterns that these two proteins exhibit. Measurements of binding, chemotaxis and calcium influx conducted with hMIP-1 alpha and hMIP-1 beta chimeric proteins and mutants show that two amino acids (K-37 and L-43) are important in the binding and signaling of hMIP-1 alpha through CKR1. Furthermore, we also show that mutations of the three charged amino acids at the C-terminus of hMIP-1 alpha and hMIP-1 beta (amino acids 61, 65 and 67), do not adversely affect the binding to THP-1 cells.