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Biotech / Medical : Ligand (LGND) Breakout! -- Ignore unavailable to you. Want to Upgrade?

To: Lyn Benson who wrote (22663)	6/24/1998 7:47:00 PM
From: Robert L. Ray	Read Replies (1) \| Respond to of 32384

Thanx, but TUNE is the symbol for TCI Music. I sorta doubt they're also into selling rats:) I also entered the word Tune into the company lookup on Yahoo but didn't come up with much of note.. Any other ideas?

To: Lyn Benson who wrote (22663)	6/25/1998 7:21:00 AM
From: Henry Niman	Read Replies (2) \| Respond to of 32384

It looks like LGND has many presentations at the Endocrine Society Conference. Here's an anti-androgen abstract scheduled for presentation tomorrow: P3-126 EFFECTS OF THE NOVEL NON-STEROIDAL ANTI-ANDROGEN, LG120907, IN SUPPRESSION OF THE R3327H DUNNING RAT PROSTATE TUMOR Xiao-Ning Wang1, Chau Nguyen1, Rene Prudente1, Murriel Wagoner1, William Hunter1, William T Schrader1, Marco M Gottardis1, 1Depts of Endocrine Research and Pharmacology, Ligand Pharmaceuticals Inc., San Diego, CA, Non-steroidal anti-androgens, such as flutamide and bicalutamide, have been used as standard agents for the adjuvant treatment of advanced prostate cancer. Here we describe the characterization of a novel anti-androgen (AA) pharmacophore, LG120907, which is active in the R3327H Dunning prostate tumor model. In initial tests of prostate organ weight suppression in intact rats the IC50's of LG120907, flutamide and bicalutamide were 20, 20 and 5mg/kg, respectively. Chronic administration of LG120907 in tumor-bearing Copenhagen male rats (20mg/kg oral daily dosing for 14 weeks) significantly retarded the growth of tumors down to 17% of the tumor volume of vehicle-treated animals. LG120907, flutamide and bicalutamide were all equally efficacious in inhibiting tumor growth in this model. In these same animals, we also evaluated increases in serum gonadotropin and sex hormone concentrations. LG120907 and bicalutamide increased serum LH only 2-fold compared to vehicle-treated animals. By comparison, flutamide increased serum LH 4-fold. No increases in serum testosterone concentrations were noted in LG120907-treated animals compared to flutamide-treated (10-fold increase) and bicalutamide-treated (3-fold increase) animals. Finally, tumor-bearing male animals from the Dunning studies were mated to virgin female rats after 9-14 weeks of therapy. Pregnancy rates for females mated with vehicle-, LG120907-, bicalutamide- or flutamide-treated males were 75%, 80%, 30% and 0%, respectively. At term, 75% of females in vehicle- and LG120907-mated groups delivered pups, compared to only 23% of females mated to bicalutamide-treated males. The average litter size was greater in vehicle-treated and LG120907-treated animals (14.0 and 13.9 pups/litter, respectively) compared to the bicalutamide-male mated females (8.8 pups/litter). These studies indicate that LG120907 can be used as a template for developing anti-androgen pharmacophores which avoid adverse CNS and fertility side-effects for the chronic treatment of malignant or benign disease. Presentation Date, Time, Room: 26-Jun-98, 11:00A, CC-Exhibit Hall L=Special Session, N=Nurse Symposium, OR=Oral, P=Poster, S=Symposium

To: Lyn Benson who wrote (22663)	6/25/1998 7:37:00 AM
From: Henry Niman	Respond to of 32384

Tomorrow they will present more on their designer androgens: S39-2 NOVEL LIGANDS TARGETING THE ANDROGEN RECEPTOR L G Hamann1, J P Edwards1, R I Higuchi1, X N Wang2, M M Gottardis3, K B Marschke4, W T Schrader3, T K Jones1, 1Medicinal Chemistry, , , , 2Pharmacology, , , , 3Endocrine Research, , , , 4New Leads Discovery, Ligand Pharmaceuticals, San Diego, CA, In connection with our program aimed at discovery of androgen receptor (AR) modulators with high tissue specificity, a molecular approach has been utilized to identify compounds which have the potential to provide unique clinical opportunities for the treatment of prostate cancer, benign prostatic hypertrophy, alopecia, hirsutism, cachexia, and as male hormone replacement therapy, without commonly observed side-effects attributable to poor receptor or tissue selectivities. A novel non-steroidal AR pharmacophore has been discovered using mammalian cell-based cotransfection assays, and investigations into the structure-activity relationships surrounding this series of molecules has resulted in the identification of two distinct sub-classes of compounds possessing AR antagonist and agonist activities respectively. Continued intensive preclinical studies of this pharmacophore, structurally characterized by a linear tricyclic 1,2,3,4-tetrahydro-8-pyridono[5,6-g]quinoline core, has led to the discovery of highly efficacious compounds with attractive properties. Compounds from the AR antagonist series inhibit AR-mediated reporter gene expression in the presence of DHT, and bind to AR as potently or better than any known AR antagonists. Several analogues are also highly efficacious in classic rodent models of AR antagonism, inhibiting growth of rat ventral prostate, seminal vesicles, and levator ani muscle without accompanying increases in serum gonadotropin and testosterone levels, as is commonly observed with other known antiandrogens. Additionally, some of the most potent compounds have further demonstrated high efficacy inhibiting xenograft tumor growth in the Dunning rat model. Unlike other known antiandrogenic agents, this structural class has very high specificity for the AR, high peripheral tissue selectivity, and maintains potent in vitro antagonist activity when screened against an AR mutant commonly found in hormone-refractory prostate cancer patients. Compounds from the AR agonist series stimulate reporter gene expression in CV-1 and MDA MB-453 cells, and bind to hAR with low nanomolar affinity. Presentation Date, Time, Room: 26-Jun-98, 3:45P, CC-43 L=Special Session, N=Nurse Symposium, OR=Oral, P=Poster, S=Symposium

To: Lyn Benson who wrote (22663)	6/25/1998 7:52:00 AM
From: Henry Niman	Respond to of 32384

Yesterday they presented on Designer Estrogens: OR10-2 ESTROGEN RECEPTOR b ACTIVATES THE HUMAN RAR-ALPHA-1 PROMOTER IN RESPONSE TO TAMOXIFEN AND OTHER ER ANTAGONISTS, BUT NOT IN RESPONSE TO ESTROGEN A. Zou1, P. Fitzgerald1, K. B. Marschke1, E. A. Allegretto1, 1, Ligand Pharmaceuticals, San Diego, CA, Estrogen receptor beta (hERb), which was cloned from human testes RNA using RT-PCR, shared 100% identity with the published sequence and was constructed into a mammalian expression vector. hERa or hERb expression vector transfected into Hep G2 or COS1 cells each respond to estrogen to increase transcription from an ERE-driven reporter vector with similar fold induction through a classical mechanism involving direct binding of the receptors to DNA. RARa RNA and protein are known to be induced by estrogen in various ERa-positive breast cancer cell lines. We have shown previously that estrogen stimulates the human retinoic acid receptor alpha 1 (hRARa-1) promoter through nonclassical EREs by a mechanism that is ERa dependent, but that does not involve direct binding of the receptor to DNA. We show here that in contrast to hERa, hERb was unable to induce luciferase activity driven by the (hRARa1) promoter in response to estrogen. Interestingly, while hERb does not mediate stimulation of this promoter in response to estrogen, it does elicit transcriptional activation in the presence of 4-hydroxytamoxifen (4-OH-Tam). 4-OH-Tam acts as an agonist via ERb and estrogen has no activity alone, but estrogen is able to completely antagonize the tamoxifen activation. This induction by ERb is also independent of direct binding to DNA, however, the site of action of hERb within the RARa promoter is different than the sites of hERa activity. While hERa was shown to act through two estrogen-responsive sequences within the promoter, hERb acts at only the 3' ERE in response to 4-OH-Tam. Other ER antagonists including raloxifene, ICI164,384 and ICI182,780 also act as agonists via the hRARa1 promoter. Additionally, the progesterone receptor (PR) antagonist RU486, although it does not bind well to ERa or ERb in vitro, acts as an antagonist via hERa or hERb on an ERE-driven reporter in COS-1 or Hep G2 cells which do not contain PR, presumably through metabolism to an ER-active compound. Interestingly, RU486 acts as an agonist through ERb to induce the hRARa1 promoter in Hep G2 cells. The biological relevance of these results is currently being explored by testing the ability of tamoxifen and other ER antagonists to increase RARa RNA and protein levels in breast cancer cells expressing only ERb and not ERa. These findings may have ramifications in breast cancer treatment regimens utilizing tamoxifen or other ER antagonists. Presentation Date, Time, Room: 24-Jun-98, 1:00P, CC-26 L=Special Session, N=Nurse Symposium, OR=Oral, P=Poster, S=Symposium

To: Lyn Benson who wrote (22663)	6/25/1998 7:56:00 AM
From: Henry Niman	Respond to of 32384

Today they will talk about monoclonals that can monitor phosporylation of serine residues on progesterone receptors: OR14-3 MONOCLONAL ANTIBODIES WHICH RECOGNIZE SPECIFIC PHOSPHORYLATIONS OF HUMAN PROGESTERONE RECEPTOR D L Clemm1, L Sherman2, D P Edwards2, N L Weigel3, W T Schrader1, 1Endocrine Research, Ligand Pharmaceuticals, San Diego, CA, 2Dept. of Pathology, University of Colorado, Denver, CO, 3Dept. of Cell Biology, Balyor College of Medicine, Houston, TX, Antibodies which recognize phosphotyrosine have been invaluable in studying post-translational modification. This method has not been available for the nuclear receptors, which are largely phosphorylated at serines. Hence phosphorylation studies of these receptors have involved laborious 32P experiments. Now we have developed monoclonal antibodies which recognize oligopeptides in which the serine residue is phosphorylated. By using two different phosphopeptides as antigens (VLPRGLSPARQLL and PMAPGRSPLATTV), monoclonal antibodies #1154, and #608 have been developed which recognize specifically the human progesterone receptors when phosphorylated at Ser190 and Ser294 respectively. Competition by these phosphopeptides in excess blocks reaction of the antibody with hPR in Western protein blotting experiments whereas the non-phosphorylated peptides have no effect. We used antibody 1154 in Western blotting of SDS-PAGE gels to measure the phosphorylation state of hPR Ser190 in human breast tumor T47D cells. Both isoforms hPRB and hPRA are minimally phosphorylated at Ser190 in the absence of hormone. The degree of phosphorylation is approximately the same for both forms, consistent with earlier reports. Following addition of 100 nM of either progestin agonist R5020 or medroxyprogesterone acetate, phosphorylation intensity of both isoforms increased concomitantly by 15 minutes and rose about 3 to 5-fold by 60 min. The anti-progestin RU38486 showed a similar pattern, confirming that modification of the Ser190 site can be caused by either agonists or antagonists. All three hormones showed a dose-dependent increase in phosphorylation, with band intensities increasing dramatically between 10-9 to 10-8 M. Phosphorylation at Ser345 causes a retarded migration of the proteins. We used the changes in mobility of the phosphoSer190-containing bands to assess the relative rates of phosphorylation at Ser190 and Ser345. The upshift was noticeable only after 30 min. and was nearly complete by 60 min. Thus Ser190 phosphorylation precedes Ser345 . We have tested other hormonal modulators of PR. Pure antagonist ZK98299 caused increased anti-phosphoSer190 band intensity, but the band did not upshift. Thus phosphorylation occurred at Ser190but not Ser345. These findings confirm the utility of this method for receptor phosphorylation studies. Presentation Date, Time, Room: 25-Jun-98, 1:00P, CC-24 L=Special Session, N=Nurse Symposium, OR=Oral, P=Poster, S=Symposium

To: Lyn Benson who wrote (22663)	6/25/1998 8:01:00 AM
From: Henry Niman	Respond to of 32384

This morning they will also discuss their G-CSF mimic: S18-2 JAK/STATs AS DRUG DISCOVERY TARGETS P Lamb1, S Tian1, A King2, S Miller1, L Kessler1, J Luengo2, L Averill2, R Johnson2, J Gleason2, L Pelus2, S Dillon2, J Rosen1, 1, Ligand Pharmaceuticals, San Diego, CA, 2, SmithKline Beecham Pharmaceuticals, Collegeville, PA, Binding of cytokines to specific cell surface receptors results in activation of signal transduction pathways that regulate gene expression in the nucleus. Central to this process are a subfamily of protein tyrosine kinases known as JAKs, and a family of latent transcription factors termed STATs. JAK kinases associate with the cytoplasmic face of cytokine receptors and become activated upon receptor oligomerization. The activated JAKs phosphorylate a variety of signaling molecules including the STATs, which subsequently dimerize and translocate to the nucleus, where they bind to specific DNA sequences located in the promoters of cytokine responsive genes. The rapid activation of JAK/STAT signaling proteins by cytokines provides the opportunity to develop high throughput screens for small molecule cytokine modulators. Using a cell-based screen that detects activated STATs, we have identified a non-peptidyl small molecule capable of activating granulocyte-colony stimulating factor (G-CSF) signal transduction pathways. Like G-CSF, this compound induces tyrosine phosphorylation of multiple signaling proteins, including JAKs and STATs, and activates a panel of G-CSF early response genes. In longer term assays, the compound supports the proliferation of a G-CSF-dependent cell line, and stimulates primary murine bone marrow cells to form granulocytic colonies in vitro. Remarkably, it also elevates peripheral blood neutrophil counts in vivo, with an efficacy similar to recombinant G-CSF. The extracellular domain of the murine G-CSF receptor is required for compound activity, suggesting that the compound acts by oligomerizing receptor chains. The identification of this compound provides proof of principle for drug discovery using JAK/STAT based assays, and shows for the first time that a small non-peptidyl molecule can trigger the selective activation of a large protein hormone receptor. These findings may lead to the development of orally available cytokine mimics. Presentation Date, Time, Room: 25-Jun-98, 9:30A, CC-24 L=Special Session, N=Nurse Symposium, OR=Oral, P=Poster, S=Symposium

To: Lyn Benson who wrote (22663)	6/25/1998 8:13:00 AM
From: Henry Niman	Respond to of 32384

They will also discuss effects of high dose Targretin on TSH in CTCL patients: OR35-6 THYROID AXIS ALTERATIONS IN CTCL PATIENTS RECEIVING AN RXR-SELECTIVE LIGAND S I Sherman1, J Gopal1, A C Chiu1, K Whaley2, P Nowlakha2, J Truglia3, R Yocum3, M Duvic2, 1Sect. of Endocrine Neoplasia and Hormonal Disorders, Univ. of Texas M.D. Anderson Cancer Center, Houston, TX, 2Sect. of Dermatology, Univ. of Texas M.D. Anderson Cancer Center, Houston, TX, 3, Ligand Pharmaceuticals, San Diego, CA, , In an interim analysis of multicenter phase 2-3 trials of oral Targretin (a synthetic RXR-selective retinoid analogue) in patients with cutaneous T-cell lymphoma (CTCL), 11% (8/76) of patients had mild baseline elevations of serum TSH. Thyroid testing in 24 of 31 patients enrolled at one study center revealed abnormally low TSH levels during treatment with Targretin, <Picture>300 mg/M2/d (lower doses in 3 patients). These 24 patients (12 men, 12 women; mean age 67 yrs; TNM stage IB-III) had a baseline log mean TSH of 2.27 mU/L that fell to a nadir of 0.05 (P<0.0001), whereas the free and/or total T4 declined below normal in 22 patients. Neither a baseline elevated TSH nor detectable antithyroid antibodies (found in 5 of 21 patients tested) was associated with subsequent hypothyrotropinemia. TRH stimulation performed in 2 patients on Targretin therapy increased the TSH level 7- and 11-fold, respectively. Following initiation of Targretin therapy, elicited hypothyroid symptoms included cold intolerance (11 patients), fatigue (10), impaired cognition, constipation, and depression (3 each) muscle cramps, hoarseness, and worsening hearing acuity (2 each), and resolution of palpitations associated with chronic atrial fibrillation (1). Physical findings included delayed deep tendon reflex relaxation (4), nonpitting edema (3), and decreased strength (1). In patients whose Targretin was interrupted, TSH and T4 levels recovered from suppression while off drug and were subsequently suppressed with restarting retinoid therapy. Some clinical improvement was noted following initiation of L-thyroxine therapy. In one patient treated with L-thyroxine, 0.175 mg, a doubling of her T4 level and further suppression of TSH was seen when she stopped Targretin. In summary, biochemical evidence of central hypothyroidism was frequently observed in CTCL patients treated with high-dose Targretin, though not all patients demonstrated clinical manifestations. Although the primary mechanism is likely reversible TSH suppression by the RXR-selective ligand, additional effects on thyroid hormone secretion and/or metabolism cannot be ruled out. These observations in CTCL patients contrast with the cumulative experience with oral Targretin therapy in 192 patients with other malignancies (of whom only 1.6% were reported to develop hypothyroidism during therapy), suggesting that the thyroid axis in CTCL patients may be particularly susceptible to the effects of an RXR-selective ligand. Presentation Date, Time, Room: 27-Jun-98, 11:00A, CC-43 L=Special Session, N=Nurse Symposium, OR=Oral, P=Poster, S=Symposium

To: Lyn Benson who wrote (22663)	6/25/1998 8:26:00 AM
From: Henry Niman	Respond to of 32384

They will also be presenting on FSH in collaboration with AHP: P3-4 DUAL ACTIVITY OF INSECT CELL-PRODUCED RECOMBINANT FOLLICLE-STIMULATING HORMONE IN VIVO B J Arey1, P E Stevis1, E S Shen1, E H Meade1, K Ghosh2, F J Lopez3, 1Women's Health Research Institute, Wyeth-Ayerst Research, Radnor, PA, 2Biometrics Department, Wyeth-Ayerst Research, Radnor, PA, 3, Ligand Pharmaceuticals, San Diego, CA, , The gonadotropin FSH is secreted from the anterior pituitary gland as a series of isoforms differing in glycosylation pattern and possessing different physico-chemical as well as biological properties. We have recently shown that underglycosylation of FSH confers promiscuous G-protein coupling of the FSH receptor that depends upon the degree of activation of the receptor-signal transduction mechanism. Therefore, this mechanism could represent a means whereby the gonad may respond in a different manner to differently glycosylated isoforms of FSH. To investigate the gonadal response to alternatively glycosylated variants of FSH in vivo, immature female rats were treated with either purified human FSH (phFSH), insect-cell expressed hFSH (BV-hFSH) or both. Administration of phFSH to immature rats induced a sigmoidal dose-dependent increase in ovarian and uterine wet-weight. This is consistent with an FSH-dependent increase in follicular development. However, BV-hFSH produced a biphasic dose-dependent elevation, suggesting that at high doses the receptor recruited an inhibitory signaling pathway as shown previously using primary cultures of granulosa cells. Furthermore, BV-hFSH acted as a potent antagonist in rats treated with the ED80 of phFSH. Taken together with earlier in vitro studies, we hypothesize that the gonad in vivo has the capability to respond to differing circulating isoforms of FSH in a pleiotropic manner. In addition, FSH isoforms are capable of activation of alternative signaling pathways resulting in diverse biological effects. Since under near-maximal stimulation conditions (ED80 phFSH) BV-hFSH behaved as a complete antagonist of FSH in vivo, the relative proportion of underglycosylated forms of FSH in the complete FSH signal to the gonad may represent a protective mechanism for ovarian hyperstimulation. Moreover, relative increases of underglycosylated forms of FSH may play a role in infertility as reported in earlier studies. Presentation Date, Time, Room: 26-Jun-98, 11:00A, CC-Exhibit Hall L=Special Session, N=Nurse Symposium, OR=Oral, P=Poster, S=Symposium

To: Lyn Benson who wrote (22663)	6/25/1998 8:39:00 AM
From: Henry Niman	Respond to of 32384

Some of LGND's former employees will also be presenting. Here's an abstract by Donald McDonnell: OR14-2 DIFFERENTIAL INTERACTION OF THE METHOXYCHLOR METABOLITE, HPTE, WITH ESTROGEN RECEPTORS a AND b Kevin W. Gaido1, Linda S. Leonard1, Susan C. Maness1, Donald P. McDonnell2, Julie Galluzzo2, Brad Seville3, Stephen Safe3, 1, Chemical Industry Institute of Toxicology, Research Triangle Park, NC, 2Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, 3Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX, The recent identification of a second estrogen receptor, the estrogen receptor b has prompted intensive investigation into the role of this receptor in estrogenic responses. Estrogen receptor a and b share many similarities with respect to ligand binding affinities and gene expression regulation, although there are some differences in their transcriptional activities and tissue localization. We compared the activity at both estrogen receptor a and b of 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), an estrogenic metabolite of the organochlorine pesticide methoxychlor. HepG2 human hepatoma cells were co-transfected with either the human or rat estrogen receptor a or b together with an estrogen-responsive luciferase reporter gene. HPTE was a complete agonist with both the human and rat estrogen receptor a with an EC50 of approximately 5x10-8 M with the human ER-a and 1x10-8 M with the rat ER-a. The EC50 for estradiol was approximately 4x10-9 M and 1x10-9 M for the human and rat ER-a, respectively. In contrast, HPTE had minimal agonist activity with either the human or rat ER-b. In the presence of a maximally inducing dose of estradiol, HPTE almost completely abolished ER-b activity. Moreover, similar results were observed in other cell lines. Our results are the first to demonstrate a clear difference in activity of a single ligand between the two estrogen receptors and may lead not only to a better understanding of the mechanism of methoxychlor toxicity but also to the development of estrogen receptor a and b specific pharmaceutical agents. Presentation Date, Time, Room: 25-Jun-98, 1:00P, CC-24 L=Special Session, N=Nurse Symposium, OR=Oral, P=Poster, S=Symposium

To: Lyn Benson who wrote (22663)	6/25/1998 8:41:00 AM
From: Henry Niman	Respond to of 32384

Here's another one by McDonnell: OR40-3 ESTROGEN-INDUCED ACTIVATION OF MAPK IS ESTROGEN RECEPTOR-DEPENDENT AND REQUIRES MOBILIZATION OF INTRACELLULAR CALCIUM STORES Teresa Improta-Brears1, A. Richard Whorton1, Donald P. McDonnell1, 1Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, , Estrogens and growth factors such as EGF are known to act as mitogens promoting proliferation in breast cells and in cells of the reproductive tract. Although the mechanism by which estrogen facilitates mitogenesis has remained elusive, several studies have shown that there is cross-talk between the EGF and estrogen mediated signaling pathways, indicating some commonality in the mechanism of action of these agents. Indeed, several groups have recently demonstrated that 17-b-estradiol (E2) causes a rapid activation of MARK in mammalian cells, an event which does not involve transcription or new protein synthesis. Although the physiological relevance of MAPK activation by estrogen has yet to be proven, it does provide a potential mechanism by which estrogens can stimulate cell proliferation. We have used a pharmacological approach to dissect this novel pathway in MCF-7 breast cancer cells, with a view to understanding its role in the cellular response to estrogens and antiestrogens. These studies have revealed that activation of MAPK in response to E2 requires the classical Estrogen Receptor (ER) but occurs independently of Raf activation. Using an antibody specifically recognizing activated MAPK, however, we were able to demonstrate that activation of this enzyme by estrogen is preceded by a rapid increase in cytosolic calcium. The activity of estrogen in this assay could be mimicked by Thapsigargin, a drug known to mobilize the intracellular Ca2+ pool. Furthermore, the involvement of intracellular calcium is supported by the finding that the presence of EGTA and Ca2+/free medium did not affect the activation of MAPK in response to E2, and additionally that this response was abolished by addition of the intracellular calcium chelator BAPTA. Cumulatively, these data indicate that ER, in addition to functioning as a transcription factor, is also involved, by non-genomic mechanisms, in Ca2+ mediated and MAPK signaling pathways. Presentation Date, Time, Room: 27-Jun-98, 11:00A, CC-26 L=Special Session, N=Nurse Symposium, OR=Oral, P=Poster, S=Symposium

To: Lyn Benson who wrote (22663)	6/25/1998 8:42:00 AM
From: Henry Niman	Read Replies (1) \| Respond to of 32384

Here's a third abstract by McDonnell: OR48-3 HORMONE-DEPENDENT INTERACTIONS BETWEEN THE AMINO- AND CARBOXYL-TERMINAL REGIONS OF PROGESTERONE RECEPTOR DETECTED IN VITRO AND IN VIVO M J Tetel1, P H Giangrande2, D P McDonnell2, D P Edwards1, 1Pathology Dept., U. of Colorado HSC, Denver, CO, 2Dept of Pharmacology, Duke U. Medical Center, Durham, NC, Human progesterone receptors (PR) exists as two isoforms, PR-B and the aminoterminally truncated PR-A. PR-A can function as a transcriptional repressor of PR-B and other steroid receptors in a cell and promoter specific manner. In the present study, we provide in vitro and in vivo evidence of interaction between the amino (N)- and carboxyl (C)-terminal regions of PR that may contribute to the functional differences of the two receptor isoforms. For in vitro studies, separately expressed N and C-terminal domains of PR were used in a pull down assay to detect and quantify interactions between nonfusion and polyhistidine-tagged proteins immobilized to a metal resin. In the presence of the progestin agonist R5020, the N-terminal domains of PR-B (BN, aa 1-535) and PR-A (AN, aa 165-535) physically interacted to a similar extent with the C-terminal ligand binding domain plus hinge sequences (hLBD, aa 634-933). N-C terminal interactions did not occur when the hLBD was unliganded or bound to the antagonist RU486, indicating these interactions are agonist-dependent. Addition of nuclear receptor co-activators (SRC-1 and CBP) increased hLBD association with N-terminal domains, suggesting these interactions are indirect. A mammalian two-hybrid assay was used to investigate N-C terminal interactions in vivo using the hLBD, BN and AN fused to either the GAL4 DBD or the VP16 transactivation domain. Consistent with the in vitro binding results, interactions between N and C terminal fusion proteins, as detected by reporter gene induction, occurred in a hormone-dependent fashion with antagonist decreasing these interactions. However, in contrast to the in vitro results, in Hela cells hLBD interacted with BN more efficiently than with AN, while in HepG2 cells hLBD associated more efficiently with AN than with BN. Interestingly, these cell-type specific differences correlate with the findings that PR-A in Hela cells is transcriptionally inactive and able to transrepress, while in HepG2 cells PR-A is transcriptionally active and unable to repress. In conclusion, these in vivo and in vitro data indicate that cell-type specific factor(s) mediate PR N-C domain interactions and may contribute to the functional differences between PR-A and PR-B. Presentation Date, Time, Room: 27-Jun-98, 1:30P, CC-36 L=Special Session, N=Nurse Symposium, OR=Oral, P=Poster, S=Symposium