Subtype-specific residues involved in ligand activation of the endothelial differentiation gene family lysophosphatidic acid receptors.
Lysophosphatidic acid (LPA) is a ligand for three endothelial differentiation gene family G protein-coupled receptors, LPA(1-3). We performed computational modeling-guided mutagenesis of conserved residues in transmembrane domains 3, 4, 5, and 7 of LPA(1-3) predicted to interact with the glycerophosphate motif of LPA C18:1. The mutants were expressed in RH7777 cells, and the efficacy (E(max)) and potency (EC(50)) of LPA-elicited Ca(2+) transients were measured. Mutation to alanine of R3.28 universally decreased both the efficacy and potency in LPA(1-3) and eliminated strong ionic interactions in the modeled LPA complexes. The alanine mutation at Q3.29 decreased modeled interactions and activation in LPA(1) and LPA(2) more than in LPA(3). The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA(1) and LPA(2) but reduced the activation and modeled interactions of LPA(3). The R5.38A mutant of LPA(2) and R5.38N mutant of LPA(3) showed diminished activation by LPA; however, in LPA(1) the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA(2), K7.36A decreased the potency of LPA; in LPA(1) this same mutation increased the E(max). In LPA(3), R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC(50) in response to LPA 10-fold. In LPA(1-3), the mutation Q3.29E caused a modest increase in EC(50) in response to LPA but caused the LPA receptors to become more responsive to sphingosine 1-phosphate (S1P). Surprisingly micromolar concentrations of S1P activated the wild type LPA(2) and LPA(3) receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors.
Valentine
William J
WJ
Department of Physiology, The University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
Fells
James I
JI
Perygin
Donna H
DH
Mujahid
Sana
S
Yokoyama
Kazuaki
K
Fujiwara
Yuko
Y
Tsukahara
Ryoko
R
Van Brocklyn
James R
JR
Parrill
Abby L
AL
Tigyi
Gabor
G
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D019295:Computational Biology; D017124:Conserved Sequence; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D008024:Ligands; D008246:Lysophospholipids / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D051381:Rats; D049368:Receptors, Lysophosphatidic Acid / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism
D000596:Amino Acids; D008024:Ligands; D008246:Lysophospholipids; D050505:Mutant Proteins; D049368:Receptors, Lysophosphatidic Acid; C060506:sphingosine 1-phosphate; D013110:Sphingosine; C032881:lysophosphatidic acid