Identification and pharmacological characterization of the prostaglandin FP receptor and FP receptor variant complexes.
OBJECTIVE
A prostamide analogue, bimatoprost, has been shown to be effective in reducing intraocular pressure, but its precise mechanism of action remains unclear. Hence, to elucidate the molecular mechanisms of this effect of bimatoprost, we focused on pharmacologically characterizing prostaglandin FP receptor (FP) and FP receptor variant (altFP) complexes.


METHODS
FP receptor mRNA variants were identified by reverse transcription-polymerase chain reaction. The FP-altFP4 heterodimers were established in HEK293/EBNA cells co-expressing FP and altFP4 receptor variants. A fluorometric imaging plate reader was used to study Ca2+ mobilization. Upregulation of cysteine-rich angiogenic protein 61 (Cyr61) mRNA was measured by Northern blot analysis, and phosphorylation of myosin light chain (MLC) by western analysis.


RESULTS
Six splicing variants of FP receptor mRNA were identified in human ocular tissues. Immunoprecipitation confirmed that the FP receptor is dimerized with altFP4 receptors in HEK293/EBNA cells co-expressing FP and altFP4 receptors. In the studies of the kinetic profile for Ca2+ mobilization, prostaglandin F2alpha (PGF2alpha) elicited a rapid increase in intracellular Ca2+ followed by a steady state phase. In contrast, bimatoprost elicited an immediate increase in intracellular Ca2+ followed by a second phase. The prostamide antagonist, AGN211335, selectively and dose-dependently inhibited the bimatoprost-initiated second phase of Ca2+ mobilization, Cyr61 mRNA upregulation and MLC phosphorylation, but did not block the action of PGF2alpha.


CONCLUSIONS
Bimatoprost lacks effects on the FP receptor but may interact with the FP-altFP receptor heterodimer to induce alterations in second messenger signalling. Hence, FP-altFP complexes may represent the underlying basis of bimatoprost pharmacology.
Liang
Y
Y
Departments of Biological and Chemical Sciences, Allergan Inc., Irvine, CA, USA. liang_yanbin@allergan.com
Woodward
D F
DF
Guzman
V M
VM
Li
C
C
Scott
D F
DF
Wang
J W
JW
Wheeler
L A
LA
Garst
M E
ME
Landsverk
K
K
Sachs
G
G
Krauss
A H-P
AH
Cornell
C
C
Martos
J
J
Pettit
S
S
Fliri
H
H
D017398:Alternative Splicing; D000577:Amides / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000069580:Bimatoprost; D015152:Blotting, Northern; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003008:Cloprostenol / Q000031:analogs & derivatives* / Q000494:pharmacology; D055515:Cysteine-Rich Protein 61; D019281:Dimerization; D015237:Dinoprost / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005123:Eye / Q000187:drug effects / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D011982:Receptors, Prostaglandin / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection
D000577:Amides; C501848:CCN1 protein, human; D055515:Cysteine-Rich Protein 61; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D012333:RNA, Messenger; D011982:Receptors, Prostaglandin; C034671:prostaglandin F2alpha receptor; D003008:Cloprostenol; D015237:Dinoprost; D000069580:Bimatoprost; D002118:Calcium