Anti-inflammatory role of PGD2 in acute lung inflammation and therapeutic application of its signal enhancement.

We investigated the role of prostaglandin D2 (PGD2) signaling in acute lung injury (ALI), focusing on its producer-effector interaction in vivo. Administration of endotoxin increased edema and neutrophil infiltration in the WT mouse lung. Gene disruption of hematopoietic PGD synthase (H-PGDS) aggravated all of the symptoms. Experiments involving bone marrow transplantation between WT and H-PGDS-deficient mice showed that PGD2 derived from alveolar nonhematopoietic lineage cells (i.e., endothelial cells and epithelial cells) promotes vascular barrier function during the early phase (day 1), whereas neutrophil-derived PGD2 attenuates its own infiltration and cytokine expression during the later phase (day 3) of ALI. Treatment with either an agonist to the PGD2 receptor, DP, or a degradation product of PGD2, 15-deoxy-Δ(12,14)-PGJ2, exerted a therapeutic action against ALI. Data obtained from bone marrow transplantation between WT and DP-deficient mice suggest that the DP signal in alveolar endothelial cells is crucial for the anti-inflammatory reactions of PGD2. In vitro, DP agonism directly enhanced endothelial barrier formation, and 15-deoxy-Δ(12,14)-PGJ2 attenuated both neutrophil migration and cytokine expression. These observations indicate that the PGD2 signaling between alveolar endothelial/epithelial cells and infiltrating neutrophils provides anti-inflammatory effects in ALI, and suggest the therapeutic potential of these signaling enhancements.

Possible novel receptor for PGD2 on human bronchial epithelial cells.

Prostaglandin D(2) (PGD(2)), a major prostanoid produced by activated mast cells, has long been implicated in allergic diseases. Recent studies have shown that PGD(2) exerts its effects through two different G-protein-coupled receptors (GPCRs), the D-prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on T helper type-2 cells (CRTH2), expressed in various human tissues. The PGD(2)/CRTH2 system mediates the chemotaxis of eosinophils, basophils, and Th2 cells, which are involved in the induction of allergic inflammation. We have reported that normal human bronchial epithelial cells (NHBE) and epithelial cell lines (NCI-H(292)) expressed CRTH2, and PGD(2) induces production of IL-8 and GM-CSF. This review discusses the role of CRTH2/DP on epithelial cells and mentions a possible novel receptor for PGD(2).

Identification of prostanoid receptors in rabbit non-pigmented ciliary epithelial cells.

Preliminary ligand binding studies demonstrated that the membrane preparations of the rabbit nonpigmented ciliary epithelial cell line have 3H-prostaglandin E2 binding sites. The binding sites were specific for 3H-prostaglandin E2 as demonstrated by competition with unlabeled prostaglandin E2. The IC50 of prostaglandin E2 for the inhibition of 3H-prostaglandin E2 binding was 435 nM. The stimulation of adenylyl cyclase and phospholipase C by prostanoid receptor agonists, in rabbit non-pigmented ciliary epithelial cells resulted in the formation of either cyclic AMP or inositol phosphates. Prostaglandin E2 and 16-16-dimethyl prostaglandin E2 (both are EP1, EP2, EP3 and EP4 receptor agonists). 11-deoxy prostaglandin E1 (EP2, EP3 and EP4 receptor agonist), butaprost (EP2 receptor agonist), and prostaglandin D2 (DP receptor agonist) stimulated the formation of cyclic AMP in a dose-dependent manner. Maximal stimulation occurred between 1.25 and 2.5 microM for prostaglandin E2 and 16,16-dimethyl prostaglandin E2 and between 10 and 20 microM for 11-deoxy prostaglandin E1 and prostaglandin D2. Prostaglandin E2 and 16,16-dimethyl prostaglandin E2 were more potent (EC50 of 0.25 microM and 0.42 microM respectively) than 11-deoxy prostaglandin E1, butaprost or prostaglandin D2. The formation of cyclic AMP by prostaglandin D2 was inhibited by BW868C, a highly selective DP receptor antagonist. 17-phenyl trinor prostaglandin E2, prostaglandin F2 alpha and U46619, the EP1, FP and TP receptor agonists, respectively stimulated phospholipase C (as measured by the formation of total inositol phosphates) in a dose-dependent manner. The agonists 11-deoxy prostaglandin E1 and butaprost coupled to adenylyl cyclase via guanine nucleotide binding protein, G8, did not increase the turnover of inositol phosphates. The results of the present study suggest that rabbit non-pigmented ciliary epithelial cells express EP1, EP2, DP, FP and TP receptors.

Human chromosome 1 localization of the gene for a prostaglandin F2alpha receptor negative regulatory protein.

A protein that copurifies with the bovine prostaglandin F2alpha (FP) receptor has been isolated and the corresponding rat cDNA has been cloned. Transfection experiments suggest that this protein inhibits binding of [3H]prostaglandin F2alpha ([3H]PGF2alpha) to FP. Histologically, this protein (FP regulatory protein or FPRP) shows a distribution coinciding well with those cells and tissues that respond to PGF2alpha. A portion of the 3' untranslated region of the human homolog to fprp was subcloned, sequenced, and oligonucleotide primers chosen that allow polymerase chain reaction (PCR) amplification specifically of the human fprp sequence. These primers were then used in a PCR-based mapping-protocol. The human fprp gene was first socalized through human/rodent somatic cell hybrids to human chromosome 1 (100% concordance), and further through yeast artificial chromosome (YAC) pools to region 1p13.1-q21.3 (level 1 mapping). In view of the specific histologic localization of this negative regulator, possible pathological conditions are mentioned that may cosegrepate with this chromosomal region.

The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and northern blot analysis.

A functional cDNA clone for the mouse prostaglandin (PG) E receptor EP2 subtype was isolated from a mouse cDNA library. The mouse EP2 receptor consists of 362 amino acid residues with seven putative transmembrane domains. [3H]PGE2 bound specifically to the membrane of Chinese hamster ovary cells stably expressing the cloned receptor. This binding was displaced by unlabeled prostanoids in the order of PGE2 = PGE1 >> iloprost, a stable PGI2 agonist > PGF2 alpha > PGD2. Binding was also inhibited by butaprost (an EP2 agonist) and to a lesser extent by M&B 28767 (an EP3 agonist), but not by sulprostone (an EP1 and EP3 agonist) or SC-19220 (an EP1 antagonist). PGE2 and butaprost increased the cAMP level in the Chinese hamster ovary cells in a concentration-dependent manner. Northern blot analysis revealed that EP2 mRNA is expressed most abundantly in the uterus, followed by the spleen, lung, thymus, ileum, liver, and stomach.

Actions of the E2-isoprostane, 8-ISO-PGE2, on the platelet thromboxane/endoperoxide receptor in humans and rats: additional evidence for the existence of a unique isoprostane receptor.

D2/E2-isoprostanes, are a recently discovered series of novel prostaglandin-like compounds that are produced in vivo as products of free radical-catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase enzyme. One of the E-ring compounds expected to be produced in abundance by this mechanism, 8-iso-prostaglandin E2 (8-iso-PGE2), is a potent renal vasoconstrictor in the rat, and this effect can be abrogated by the thromboxane/endoperoxide (TxA2/PGH2) receptor antagonist SQ29548, suggesting that 8-iso-PGE2 exerts these effects by interaction with this receptor in the vasculature. Nonetheless, it has recently been suggested that 8-iso-PGE2 induces vasoconstriction by interaction with a unique receptor similar to, but distinct from, the TxA2/PGH2 receptor. Because this issue has not been resolved, we carried out studies to further examine the interaction of this compound with the TxA2/PGH2 receptor on human and rat platelets. Only at concentrations of 10(-5) M or greater did 8-iso-PGE2 induce human platelet aggregation. The aggregation was unaffected by indomethacin but was inhibited by the TxA2/PGH2 receptor antagonist SQ29548. Conversely, 8-iso-PGE2 inhibited the thromboxane receptor agonists U46619 (10(-6) M) and IBOP (3.3 x 10(-7) M) with an IC50 of 5 x 10(-7) M and 5 x 10(-6) M, respectively. 8-iso-PGE2 also inhibited platelet aggregation induced by arachidonic acid but not by ADP. Similarly in rat platelets, 8-iso-PGE2 alone.

Agonist-induced phosphorylation of human platelet TXA2/PGH2 receptors.

Thromboxane A2 (TXA2) and its precursor prostaglandin H2 (PGH2) stimulate platelet activation through interaction with TXA2/PGH2 receptors. We and others have shown that these receptors undergo homologous desensitization upon prolonged exposure to thromboxane A2 mimetics. Phosphorylation of receptors has previously been reported to be an important mechanism for receptor desensitization. In the present study we examined the possibility that homologous desensitization of human platelet TXA2/PGH2 receptors may involve phosphorylation. The ATP pool of human platelets was metabolically prelabeled with 32Pi and the labeled platelets were subsequently exposed for 1 and 10 min to the stable TXA2/PGH2 mimetic, U46619 (1 microM). TXA2/PGH2 receptors were purified approx. 2000-fold by affinity and wheatgerm lectin chromatography and subjected to SDS-PAGE followed by autoradiography. A phosphorylated plasma membrane glycoprotein (M(r) = 50-57 kDa) was detected with characteristics similar to the TXA2/PGH2 receptor. This glycoprotein was found to be phosphorylated in the unstimulated state but phosphorylation was increased by exposure to U46619. Phosphorylation occurred rapidly and was inhibited when platelets were preincubated with the TXA2/PGH2 receptor antagonist, SQ29548 (5 microM), before being stimulated with U46619. These results suggest that human platelet TXA2/PGH2 receptors are phosphoproteins and that the level of phosphorylation is increased during homologous desensitization.

Purification of rat brain, rabbit aorta, and human platelet thromboxane A2/prostaglandin H2 receptors by immunoaffinity chromatography employing anti-peptide and anti-receptor antibodies.

In the present study, a new polyclonal antibody (TxAb) was raised against native thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor protein. Previously developed anti-peptide antibodies (P1Ab, P2Ab) and TxAb were then used to prepare immunoaffinity columns to purify TXA2/PGH2 receptors from platelets, brain, and aorta. In platelets, SDS-polyacrylamide gel electrophoresis revealed the purification of a 55-kDa protein by each affinity column. Identification of this protein as the TXA2/PGH2 receptor was based on: 1) an identical electrophoretic mobility to authentic receptor; 2) immunoblotting of TxAb against P1Ab and P2Ab-purified protein; 3) immunoblotting of P1Ab/P2Ab against TxAb-purified protein; and 4) specific [3H]SQ29,548 binding to TxAb-purified protein. P1Ab/TxAb purification of receptors from brain revealed a major protein band at 55 kDa. Furthermore, the eluates from ligand affinity chromatography confirmed the presence of this 55-kDa protein in brain (which was immunoblotted with TxAb), and contained specific [3H]SQ29,548 binding. In addition to the 55-kDa protein, P1Ab/TxAb also purified a minor protein in brain at 52 kDa, which when concentrated, cross-blotted with TxAb and P1Ab. This finding indicates sequence homology between the 55- and 52-kDa proteins. Independent identification of brain TXA2/PGH2 receptors was provided by P2Ab/TxAb immunohistochemistry, which demonstrated specific labeling of discrete myelin-containing fiber tracts. P2Ab/TxAb purification of TXA2/PGH2 receptors from aorta also revealed a major protein band at 55 kDa and a minor band at 52 kDa. These results represent the first purification of TXA2/PGH2 receptors from either brain or aorta.

Identification of Gq as one of the G-proteins which copurify with human platelet thromboxane A2/prostaglandin H2 receptors.

The present study employed ligand affinity and immunoaffinity chromatography to isolate human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor-coupled G-proteins. Purification of TXA2/PGH2 receptors by ligand (SQ31,491)-affinity chromatography resulted in the elution of receptor binding and GTPase activity in the same fraction. GTPase activity of this fraction was enriched (6-fold) relative to solubilized platelet membranes, was stimulated (65%) by 500 nM U46619, and was blocked (74%) by 250 nM SQ29,548. Furthermore, GTP (100 microM) increased [3H]SQ29,548 receptor binding by 48%. Immunoblotting of this fraction against QL antiserum identified a 42-kDa protein as a member of the Gq family. In separate experiments, TXA2/PGH2 receptors were purified by immunoaffinity chromatography using P1Ab, P2Ab, and TxAb affinity columns. QL-immunoreactive proteins at 42 kDa were found in all three column eluates. Studies using G alpha,common antiserum (GA/1) demonstrated immunoblotting of two proteins of approximately 42 and 85 kDa in both the ligand and P2Ab affinity column fractions. On the other hand, the P1Ab and TxAb affinity column eluates contained GA/1 immunoreactivity only in the 42 kDa region. Collectively, these data identify Gq as a TXA2/PGH2 receptor-coupled G-protein and suggest the association of this receptor with additional G alpha subunits.

Cloning and expression of cDNA for a mouse EP1 subtype of prostaglandin E receptor.

A functional cDNA clone encoding a mouse EP1 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse thromboxane A2 receptor cDNA. The clone isolated encodes a protein consisting of 405 amino acid residues with putative seven-transmembrane domains. [3H]PGE2 specifically bound to the membrane of Chinese hamster ovary cells expressing this clone. The binding to the membrane was displaced with unlabeled PGs in the order of PGE2 > iloprost (a prostacyclin analogue) > PGE1 > PGF2 alpha > U-46619 (a thromboxane A2 analogue) > PGD2. The binding was also inhibited by 17-phenyl trinor PGE2 (an EP1 agonist) and sulprostone (an EP1 and EP3 agonist) but not by 11-deoxy PGE1 (an EP2 and EP3 agonist) and butaprost (an EP2 agonist). PGE2 induced a rapid increase in intracellular Ca2+ concentration in Chinese hamster ovary cells expressing the receptor. These results suggest that this receptor belongs to EP1 subtype of PGE receptor. Northern blot analysis demonstrated that the mRNA of this receptor is expressed abundantly in kidney and in a lessor amount in lung.

Stimulatory (EP1 and EP3) and inhibitory (EP2) prostaglandin E2 receptors in isolated ileal smooth muscle cells.

Isolated smooth muscle cells from the circular layer of pig and guinea-pig ileum were used to study the effect of prostaglandin E2 (PGE2) and three PGE2 receptor (EP) agonists; iloprost (EP1), butaprost (EP2) and enprostil (EP3). In pig cells, PGE2 and enprostil induced cell contraction (22.1 and 21.5% shortening of cell length, obtained at 10 nM for PGE2 and 1 nM for enprostil, respectively). Iloprost and butaprost had no contractile effect. However, the cholecystokinin octapeptide (CCK-8; 10 nM)-induced contraction was inhibited when cells were preincubated with iloprost or butaprost. In guinea-pig cells, PGE2, butaprost and iloprost induced cell contraction, whereas enprostil had no effect (23.1% for 10 nM PGE2, 22.8% for 1 nM butaprost and 22.6% for 10 nM iloprost). Preincubation with SC19220 (EP1 antagonist) inhibited the PGE2-, butaprost- and iloprost-induced contractions. When the contractile effect of PGE2, butaprost and iloprost was inhibited by addition of SC19220, these agents inhibited the cell contraction induced by CCK-8 (1 nM). Smooth muscle cells from guinea-pig and pig ileum express two PGE2 receptor subtypes that induce opposite effect. EP1 and EP3 receptors mediate cell contraction in guinea-pig and pig, respectively, whereas EP2 receptors mediate cell relaxation in both species.

Characterization by photoaffinity labelling of the human platelet thromboxane A2/prostaglandin H2 receptor: evidence for N-linked glycosylation.

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) are potent proaggregatory and vasoconstrictor lipids acting through a receptor referred to as the TXA2/PGH2 receptor. The receptor was purified using a modification of a previously described method from human platelet membranes solubilized using the detergent (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and a combination of affinity chromatography and wheat germ lectin chromatography. This procedure resulted in a 1075 +/- 375-fold purification and a specific activity of 1.45 +/- 0.55 nmol/mg protein (n = 5). Repeating these chromatography steps on this partially purified receptor resulted in a preparation with a specific activity of 21 +/- 3 nmol/mg protein (n = 5). This represents the theoretical specific activity if one assumes a molecular weight of 50,000 for the receptor. The fold purification was 11,750 +/- 1250 based on crude membranes and an overall yield of 24%. To further the characterization of this receptor, we synthesized a new radioiodinated photoaffinity probe, 7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4-azido-3-iodobenzenesulfonylamino++ + )- bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid (I-SAP-N3). [125I]l-SAP-N3 irreversibly incorporated into the purified receptor yielding a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography and indicated a molecular weight for the receptor of 50-51 kDa. The incorporation of the ligand could be inhibited by a variety of TXA2/PGH2 analogues. In addition, photoaffinity labelling was inhibited in a stereoselective manner as demonstrated by the pair of enantiomers (d)- and (l)-S145. Digestion of photoaffinity labelled receptor with N-glycosidase F demonstrated the presence of at least two N-linked glycosylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization and purification of the prostaglandin E2 receptor from cardiac sarcolemma.

A prostaglandin E2 (PGE2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [3H]PGE2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg2+, Ca2+ or of changes in buffer pH were observed on [3H]PGE2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE1 displaced over 90% of [3H]PGE2 from the CHAPS-solubilized receptor. PGD2, PGI2, PGF2 alpha and 6-keto-PGF1 alpha were not effective in displacing [3H]PGE2 from the receptor. Scatchard analysis of [3H]PGE2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE2 binding sites with Kd of 0.33 +/- 0.05 nM and 3.00 +/- 0.27 nM and Bmax of 0.5 +/- 0.04 and 2.0 +/- 0.1 pmol/mg of protein. The functional PGE2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [3H]PGE2. Unlabelled PGE1 and PGE2 displaced [3H]PGE2 from the purified receptor. Scatchard analysis of [3H]PGE2 binding to purified receptor revealed the presence of the two binding sites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M(r) of approx. 100,000. This 100-kDa was photolabelled with [3H]azido-PGE2, a photoactive derivative of PGE2. We propose that this 100-kDa protein is a cardiac PGE2 receptor.

Purification of the human blood platelet thromboxane A2/prostaglandin H2 receptor protein.

The human platelet thromboxane A2/prostaglandin H2 receptor has been purified 6100-fold to apparent homogeneity by a three-step chromatographic procedure with an overall yield of 6%. A 6-fold purification of the receptor was first achieved by chromatography of 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS)-solubilized membrane proteins from human platelets on a diethylaminoethyl (DEAE)-Sepharose column. The DEAE eluate fractions containing receptor activity were then applied to a newly developed affinity column using the cyclohexyl derivative of SQ30,741 (SQ31,491) as the immobilized ligand. Elution of the receptor from the affinity column with BM13.177 yielded a further purification of 1700-fold. An additional 4-fold receptor purification from the affinity column eluate was achieved by HPLC using GPC 500 and GPC 100 columns connected in tandem. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the HPLC eluate containing purified receptor revealed a single, distinct band with a molecular weight of 55,000. The receptor binding activity was detected with [3H]SQ29,548 using a newly developed binding assay which involved immobilization of the receptor on polyethyleneimine-treated glass fiber filters. The binding of [3H]SQ29,548 to the purified receptor was time dependent, saturable, reversible and highly specific. Unlabeled SQ29,548, BM13.505, and U46619 (but not thromboxane B2 or 6-keto prostaglandin F1 alpha) competed for [3H]SQ29,548 binding to the purified receptor in a concentration-dependent manner. Scatchard analysis of [3H]SQ29,548 binding to the purified receptor revealed the presence of a single class of high-affinity binding sites, with a Kd of 4 nM and a Bmax of 17 nmol/mg protein.

Prostaglandin E1 receptor from mouse mastocytoma P-815 cells couples to 60 kDa GTP-binding protein.

The stable [3H]prostaglandin E1 (PGE1)-bound receptor, which couples to 60 kDa GTP-binding protein, from membranes of mouse mastocytoma P-815 cells has been purified and characterized. When the membranes were preincubated with [3H]PGE1 for 60 min at 37 degrees C, the dissociation of the ligand from the receptor was remarkably decreased, even in the presence of GTP gamma S. The stable [3H]PGE1-bound receptor complex was solubilized with 6% digitonin. The solubilized [3H]PGE1 receptor was eluted with [35S]GTP gamma S bindings activity from an Ultrogel AcA44 column. The fractions containing activities of both [3H]PGE1 and [35S]GTP gamma S bindings were further purified by column chromatographies on wheat germ agglutinin (WGA)-agarose and phenyl-Sepharose CL-4B. The partially purified [3H]PGE1-bound receptor was affinity-labeled with [14C]5'-p-fluorosulfonylbenzoylguanosine and a protein with a molecular mass of 60 kDa was detected. These results suggest that the ligand-bound PGE1 receptor of P-815 cells associates with a novel GTP-binding protein with a molecular mass of 60 kDa.

Characterization of partially purified prostaglandin E2 receptor from the canine renal medulla: evidence for physical association of the receptor protein with the inhibitory guanine nucleotide-binding protein.

Prostaglandin (PG) E2 binding protein, a putative PGE2 receptor, was purified 26-fold with 0.4% recovery from canine renal outer medullary membranes solubilized with 12% digitonin with the sequential use of a Superose 12, Wheat Germ Agglutinin (WGA) Affigel 10, DEAE-5PW and Ampholine column chromatographies. The final preparation retained the binding activity specific for PGE2, but lost most of the sensitivity to guanosine-5'-(gamma-thio)triphosphate (GTP gamma S). An antibody against alpha subunit of the inhibitory guanine nucleotide-binding protein (alpha Gi)1 and alpha Gi2 or that against common sequences of alpha subunit of guanine nucleotide-binding proteins (alpha G(common)) reacted at 41 kDa protein in the sample of each step of purification, but failed to do so in the final preparation. An antibody against alpha Gi3 or alpha Go had no effect. In fact, peaks of the binding activity and immunoreactivity for alpha Gi1,2 were chromatographically separated by isoelectric focusing. Moreover, antibodies against alpha G(common) or alpha Gi1,2, but not that against alpha Gi3 and alpha Go, precipitated PGE2 binding activity in the active fractions of WGA-Affigel 10 column chromatography. These results suggest that the PGE2 receptor is an acidic glycoprotein and that Gi1 or Gi2 is physically associated with the PGE2 receptor and dissociates from the receptor protein during purification procedures.

Identification and purification of a bovine corpora luteal membrane glycoprotein with [3H]prostaglandin F2-alpha binding properties.

A bovine corpora luteal membrane glycoprotein which coelutes from multiple chromatographic procedures with bound tritiated prostaglandin F2a ([3H]PGF2 alpha) has been identified and purified to homogeneity. The properties of this molecule include: an apparent molecular mass by polyacrylamide gel electrophoresis (PAGE) of 135 kD; glycosylation which resists endoglycosidases D and H but is susceptible to cleavage by the exoglycosidase sialidase; binding of the molecule to Wheat Germ Agglutinin Sepharose but not to Concanavalin A Sepharose or Soybean Agglutinin Sepharose; migration on O'Farrell 2-D PAGE (pI 3-10) to the acidic side of the gel; binding to DEAE-Cellulose at pH 7.5 which can be displaced with NaCl at concentrations above approximately 100 mM; and, when solubilized with Triton X-100, binding to Phenyl-Sepharose or Octyl-Sepharose columns. Lastly, a rabbit polyclonal antibody against this [3H]PGF2 alpha binding protein has been made which allows both Western blotting of the 135 kD protein as well as immunohistochemical staining of ovarian tissue in a manner expected from previous binding studies. Problems associated with membrane solubilization of the receptor and receptor renaturation are discussed.

Photolabelling of the prostaglandin E2 receptor in cardiac sarcolemmal vesicles.

A [3H]azidophenacyl ester of PGE2 ([3H]azido-PGE2) was synthesized and used to photoaffinity label the protein component of the high affinity PGE2 binding site in cardiac sarcolemma membrane. Photolysis of the isolated cardiac sarcolemmal vesicles in the presence of [3H]azido-PGE2 resulted in the covalent labelling of a protein component that migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 100,000. Incorporation of the [3H]azido-PGE2 did not occur in the absence of photolysis. The photolabelling of the 100-kDa protein by [3H]azido-PGE2 was inhibited by excess unlabelled PGE2 and azido-PGE2. Specific binding of [3H]azido-PGE2 was displaced by excess unlabelled PGE2 or azido-PGE2, but not PGF2 alpha, 6-keto-PGF1 alpha or PGD2. These results indicate that the 100-kDa photoaffinity labelled [3H]azido-PGE2 binding protein contains the binding site for PGE2 in isolated cardiac sarcolemma membranes.