Abstract
To investigate regulatory domains of the thromboxane A2(TxA2) receptor, we constructed a truncated form of the mouse TxA2 receptor and expressed it in a mesangial cell line. The mutant receptor lacked 22 amino acids in the C-terminus including four potential phosphorylation sites. Ligand binding of mutant receptors was identical with the wild type. Stimulation with TxA2 agonist induced increases in inositol trisphosphate (IP3) generation and [Ca++]i by both wild-type and mutant receptors. However, the initial increase in IP3 generation by the mutant receptor was only ≈50% of that seen in the wild type. Exposure of wild-type receptors to TxA2 agonist caused desensitization of IP3 and calcium responses. Pretreatment with TxA2 agonist caused some desensitization of mutant receptors, but the extent of desensitization was reduced compared with the wild type. The protein kinase C inhibitor staurosporine attenuated TxA2-induced desensitization of wild-type receptors, but had little effect on TxA2-induced desensitization of mutant receptors. Pretreatment with low concentrations of the phorbol ester, phorbol 12,13-dibutyrate (100 nM), reduced subsequent responsiveness of wild-type but not mutant TxA2 receptors. In contrast, high-dose phorbol 12,13-dibutyrate (1 μM) produced a similar degree of desensitization of both receptor types. These data suggest that: 1) the C-terminus participates in coupling of the TxA2receptor to its effector systems; 2) the C-terminus contributes to agonist-specific desensitization of the TxA2 receptor; and 3) protein kinase C-induced desensitization of the TxA2receptor is complex and depends, in part, on C-terminal domains of the TxA2 receptor.
Footnotes
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Send reprint requests to: Robert F. Spurney, M.D., Box 3014, Duke University Medical Center, Durham, NC 27710.
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↵1 These studies were supported by grants from the American Heart Association (94014530) and the National Institutes of Health (R29-DK47333).
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↵2 An Established Investigator of the American Heart Association.
- Abbreviations:
- TxA2
- thromboxane A2
- Tris
- tris(hydroxymethyl)-aminomethane
- [I]BOP
- [15-(1α,2β(5Z),3α-(1E, 3S)4α)]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxa-bicyclo[2.2.1]hept-2-yl]-5-heptenoic acid
- U46619
- [(15S)-hydroxy-11α,9α-(epoxymethano)prosta-5Z,13E-dienoic acid]
- SQ29548
- ([1S-1α,2β(5Z),3β,4α))-7-(3-((2-((phenyl-amino)-carbonyl)hydrazino)methyl)-7-oxa-bicyclo-(2.2.1)heptan-2-yl)-5-heptenoic acid])
- Kd
- dissociation constant
- Bmax
- maximal number of specific binding sites
- Ki
- dissociation constant for competitive inhibitors
- PKC
- protein kinase C
- PLC
- phospholipase C
- HEPES
- 4-(-hydroxyethyl)-1-piperazineethanesulfonic acid
- HBSS
- Hank’s balanced salt solution
- KRB
- Krebs-Ringer buffer
- EGTA
- ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- PDBu
- phorbol 12, 13-dibutyrate
- G-protein
- guanine nucleotide regulatory protein
- IP1
- inositol monophosphates
- IP2
- inositol bisphosphates
- IP3
- inositol trisphosphates
- PI
- phosphoinositide
- PGE2
- prostaglandin E2
- TxB2
- thromboxane B2
- PI
- phosphatidylinositol
- [Ca++]i
- intracellular calcium level
- DMEM
- Dulbecco’s modified Eagle’s medium
- PCR
- polymerase chain reaction
- DNA
- deoxyribonucleic acid
- EDTA
- (ethylenedinitrilo)-tetraacetic acid
- fura-2AM
- fura 2 acetoxymethyl ester
- C-terminus
- carboxyl terminus
- Received February 20, 1997.
- Accepted June 2, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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