IDENTIFICATION OF TRANSMEMBRANE DOMAIN RESIDUES DETERMINANT IN THE STRUCTURE-FUNCTION RELATIONSHIP OF THE HUMAN PLATELET-ACTIVATING-FACTOR RECEPTOR BY SITE-DIRECTED MUTAGENESIS
Jl. Parent et al., IDENTIFICATION OF TRANSMEMBRANE DOMAIN RESIDUES DETERMINANT IN THE STRUCTURE-FUNCTION RELATIONSHIP OF THE HUMAN PLATELET-ACTIVATING-FACTOR RECEPTOR BY SITE-DIRECTED MUTAGENESIS, The Journal of biological chemistry, 271(38), 1996, pp. 23298-23303
Platelet-activating factor (PAF) is a potent phospholipid mediator tha
t produces a wide range of biological responses. The PAF receptor is a
member of the seven-transmembrane GTP binding regulatory protein-coup
led receptor superfamily. This receptor binds PAF with high affinity a
nd couples to multiple signaling pathways, leading to physiological re
sponses that can be inhibited by various structurally distinct PAF ant
agonists. We have used site-directed mutagenesis and functional expres
sion studies to examine the role of the Phe(97) and Phe(98) residues l
ocated in the third transmembrane helix and Asn(285) and Asp(289) of t
he seventh transmembrane helix in ligand binding and activation of the
human PAF receptor in transiently transfected COS-7 cells. The double
mutant FFGG (Phe(97) and Phe(98) mutated into Gly residues) showed a
3-4-fold decrease in affinity for PAF, but not for the specific antago
nist WEB2086, when compared with the wild-type (WT) receptor. The FFGG
mutant receptor, however, displayed normal agonist activation, sugges
ting that these two adjacent Phe residues maintain the native PAF rece
ptor conformation rather than interacting with the ligand. On the othe
r hand, substitution of Ala for Asp(289) increased the receptor affini
ty for PAF but abolished PAF-dependent inositol phosphate accumulation
; it did not affect WEB2086 binding. Substitution of Asn for Asp(289),
however, resulted in a mutant receptor with normal binding and activa
tion characteristics. When Asn(285) was mutated to Ala, the resulting
receptor was undistinguishable from the WT receptor. Surprisingly, sub
stitution of Ile for Asn(285) led to a loss of ligand binding despite
normal cell surface expression levels of this mutant, as verified by f
low cytometric analysis. Our data suggest that residues 285 and 289 ar
e determinant in the structure and activation of the PAF receptor but
not in direct ligand binding, as had been recently proposed in a PAF r
eceptor molecular model.