ALANINE EXCHANGES OF POLAR AMINO-ACIDS IN THE TRANSMEMBRANE DOMAINS OF A PLATELET-ACTIVATING-FACTOR RECEPTOR GENERATE BOTH CONSTITUTIVELY ACTIVE AND INACTIVE MUTANTS

To determine ligand-binding sites of a platelet-activating factor (PAF ) receptor, alanine-scanning mutagenesis was carried out. All 23 polar amino acids in the putative 7-transmembrane (TM) domains of a guinea pig PAF receptor were individually replaced with alanine. The ligand-b inding properties of mutant receptors were determined after transient expression in COS-7 cells, Mutants in TM II (N58A, D63A), TM III (N100 A, T101A, S104A) and TM VII (D289A) displayed higher PAF-binding affin ities than seen with the wild-type receptor. In contrast, mutants in T M V (H188A), TM VI (H248A, H249A, Q252A), and TM VII (Q276A, T278A) sh owed lower affinities. Representative mutants were then stably express ed in Chinese hamster ovary cells to observe PAF-induced cellular sign als (arachidonate release, phosphatidylinositol hydrolysis, adenylyl c yclase inhibition). An N100A mutant with the highest affinity was cons titutively active and was responsive to lyso-PAF, an inactive derivati ve of PAF. One nanomolar PAF induced no signals in low affinity mutant s, an EC(50) value for the wild-type receptor. Three histidines (His-1 88, His 248, His 249) might form a binding pocket for the phosphate gr oup of PAF, since zinc effectively inhibited ligand binding. Based on these results, a three-dimensional molecular model of PAF and its rece ptor was generated using bacteriorhodopsin as a reference protein.