MUTATIONS OF 2 ADJACENT AMINO-ACIDS GENERATE INACTIVE AND CONSTITUTIVELY ACTIVE FORMS OF THE HUMAN PLATELET-ACTIVATING-FACTOR RECEPTOR

We have mutated two residues, Ala(230) and Leu(231), in the C-terminal portion of the third intracellular loop of the human platelet-activat ing factor (PAF) receptor into Glu(230) and Arg(231), respectively. Th e Leu(231) --> Arg(231) substitution. led to two major modifications: 1) increased constitutive activity of the PAF receptor resulting in ag onist-independent production of inositol phosphates and 2) increased a ffinity of the receptor for binding PAF (agonist) but not WEB2086 (ant agonist). The L231R mutant was able to adopt at least two conformation s: (i) a higher affinity state than the corresponding state of the wil d-type receptor (WT), dependent on G protein coupling, and (ii) a low affinity state, higher than the one for the uncoupled WT receptor. The Ala(230) --> Glu(230) substitution also resulted in two major modific ations: 1) unresponsiveness in terms of phosphatidylinositol hydrolysi s in response to PAF and 2) a marked decrease in affinity of the recep tor for binding the agonist but not the antagonist. Competition bindin g studies of transient receptor expression in COS-7 cells and the inab ility of guanosine 5'-O-(3-thiotriphosphate) to modulate the decrease in affinity of a stable A230E mutant in Chinese hamster ovary cells su ggest an inherent low affinity conformation for this mutant. Alternati vely, mutation of Ala(230) to Gln(230) suggested that the residue 230 has a fundamental effect on receptor affinity and its charge is determ inant in G protein coupling of the PAF receptor. In this report, we sh ow that substitution of two immediately adjacent residues of the PAF r eceptor, Ala(230) and Leu(231), surprisingly leads to an inactive and a constitutively active phenotype, respectively. These results further support the concept of constitutively active G protein-coupled recept ors as adopting ''active'' state conformations similar to those induce d by agonist binding to WT receptors.