POINT MUTATION INCREASES A FORM OF THE NK1 RECEPTOR WITH HIGH-AFFINITY FOR NEUROKININ-A AND NEUROKININ-B AND SEPTIDE

1 The binding modalities of substance P and neurokinin A on the wild t ype and Gly(166) to-Cys mutant NK1 receptors expressed on CHO cells we re investigated in homologous and heterologous binding experiments usi ng both radiolabelled substance P and neurokinin A. 2 On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low ap parent affinity, despite its high-affinity binding constant (determine d in homologous binding experiments). The Gly(166) to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, th ereby, eliminates the discrepancy between the low apparent affinity an d the high binding constant of neurokinin A. 3 On the wild type recept or the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approxim ately the same number of sites on the mutant receptor. 4 Simultaneous mass action law analysis of binding data in which multiple radioligand s were employed in parallel demonstrated that a one-site model was una ble to accommodate all the experimental data, whereas a two-site model provided a dramatically better description. 5 These two receptor-site s display equally high affinity for substance P, while neurokinin A st rongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, w hich instead specifically alters the distribution between receptor sit es in favour of a high affinity neurokinin A binding form. 6 The low a pparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increas es the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A. 7 The binding modaliti es of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl est er) are affected similarly to neurokinin A and are better resolved int o two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affi nity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P wit h a single high affinity site that is unaffected by the mutation. 8 Th ese findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligan ds. Hence, the Gly(166) in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective f orm of the receptor.