Cloning and functional characterization of the rat stomach fundus serotonin receptor.

A DNA segment homologous to the third exons of the serotonin 1C and 2 receptor genes was isolated from a mouse genomic library. The positions of the introns flanking these exons were conserved in the three genes. To examine whether the new fragment was part of an active gene, we used a quantitative PCR protocol to analyse rat RNAs from different tissues and ages. The gene was expressed in stomach fundus at an abundance of 1 x 10(5) mRNA molecules. This tissue contracts in response to serotonin via a receptor that has previously resisted classification. We constructed a cDNA library from rat stomach fundus and isolated clones containing 2020 bp inserts with open reading frames of 465 amino acids comprising seven putative membrane-spanning regions. The protein was transiently expressed in COS cells and binding of serotonergic ligands to the membranes was analysed. The pharmacological profile resembled that described for the serotonin-stimulated contraction of the stomach fundus. After expression of this receptor in Xenopus oocytes, the application of serotonin triggered the typical chloride current which presumably results from the activation of phospholipase C. The coupling to this response system was less efficient than that of the 5-HT1C or 5-HT2 receptors.     

Ligand-dependent corepressor acts as a novel androgen receptor corepressor, inhibits prostate cancer growth, and is functionally inactivated by the Src protein kinase

The activated androgen receptor (AR) promotes prostate cancer (PCa) growth. AR antagonists repress the AR by recruitment of corepressors. Not much is known about the inactivation of AR by corepressors in the presence of agonists (androgens). Here we show that the corepressor LCoR acts as an androgen-dependent corepressor that represses human PCa growth in vivo. In line with this, progressive decrease of ligand-dependent corepressor expression was observed in the PCa TRAMP mouse model with increasing age. LCoR interacts with AR and is recruited to chromatin in an androgen-induced manner. Unexpectedly, the LXXLL motif of LCoR is dispensable for interaction with the AR. Rather, the data indicate that LCoR interacts with the AR DNA binding domain on DNA. Interestingly, the interaction of LCoR with AR is inhibited by signaling pathways that are associated with androgen-independent PCa. Here we also show that the Src kinase inactivates the corepressive function of LCoR. Interfering with endogenous Src function by a dominant negative Src mutant, the growth inhibitory activity of LCoR is enhanced in vivo in a xenograft mouse model system. Thus, our studies indicate a role of LCoR as an AR corepressor and a tumor suppressor. Further, the decreased expression or inactivation of LCoR is as an important step toward PCa carcinogenesis in vivo.     

Purification and characterization of the androgen receptor from rat ventral prostate

The androgen receptor has been purified from rat ventral prostate cytosol by a combination of differential DNA-Sepharose 4B chromatography and testosterone 17 beta-hemisuccinyl-3,3'-diaminodipropylamine-Sepharose 4B affinity chromatography. Approximately 8 micrograms of protein was obtained from 38 g of rat ventral prostate, with a yield of 24%. The receptor was purified approximately 120 000-fold. Silver nitrate staining of a sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel revealed a major polypeptide band migrating at 86 000 daltons. Affinity labeling of a partially purified receptor preparation with either 17-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one or 17 beta-hydroxy-[1,2,4,5,6,7,16,17-3H8]-5 alpha-androstan-3-one 17-(2-bromoacetate) produced a major band of radioactivity migrating at 86 000 daltons on a NaDodSO4 gel. Under nondenaturing conditions, a Mr of 85 000 was determined by gel filtration (42 A) and sucrose gradient sedimentation analysis (4.5 S). The purified receptor had an isoelectric point of 6.3 [3H]-4,5 alpha-Dihydrotestosterone, bound to the purified receptor, was displaced with 4,5 alpha-dihydrotestosterone greater than testosterone much greater than progesterone greater than 5 alpha-androstane-3 alpha, 17 beta-diol greater than 17 beta-estradiol greater than cortisol. A number of physicochemical properties of the purified receptor were similar to those of the receptor in crude cytosol.     

Purification and characterization of the untransformed androgen receptor in rat prostate

The isolation and characterization of the untransformed form of androgen receptors has not yet been successful, owing to their inherent lability as well as to their ready proteolysis. In this study, we have stabilized rat prostate androgen receptors by sodium molybdate and by rapid filtration on phosphocellulose. Proteases were inhibited by bacitracin, aprotinin, leupeptin and PMSF. Under these conditions the untransformed complex was purified approx 3000-fold, corresponding to 18% yield, by differential chromatography on DEAE cellulose and phosphocellulose gels. The partially purified receptor has the same ionic characteristics as the original untransformed receptor of crude cytosol; in addition, it possesses a Stokes' radius of 75 A, as determined by Sephacryl S-300 gel filtration, a sedimentation coefficient of 8.8S, a calculated molecular weight of 275 kDa and a friction coefficient of 1.6. The [3H]R1881 receptor complex was specific to androgens since unlabelled R1881 and dihydrotestosterone were able to completely displace bound [3H]R1881, whereas estradiol, cortisol, and triamcinolone acetonide did not compete. The purified complex was a multimer dissociable by 0.6 M KCl, resulting in a form migrating in the 4S area on sucrose density gradient. After treatment with 0.5% formaldehyde, three forms were obtained, migrating in the areas of 8-9, 5-6 and 3-4S respectively, of a sucrose density gradient containing 0.6 M KCl. This is the first step towards the purification to homogeneity of the untransformed androgen receptor.     

Molecular characterization of a functional cDNA for rat substance P receptor

This paper describes the amino acid sequence of the rat substance P receptor and its comparison with that of the rat substance K receptor on the basis of molecular cloning and sequence analysis. From a rat brain cDNA library constructed with an RNA expression vector, we identified a cDNA mixture containing a functional substance P receptor cDNA by examining electrophysiologically a receptor expression following injection of the mRNAs synthesized in vitro into Xenopus oocytes. A receptor cDNA clone was then isolated by cross-hybridization with the bovine substance K receptor cDNA. The clone was confirmed by selective binding of substance P to the cloned receptor expressed in mammalian COS cells. The deduced amino acid sequence (407 amino acid residues) possesses seven putative membrane spanning domains and shows a sequence similarity to the members of G-protein-coupled receptors. The rat substance P and substance K receptors are very similar in both size and amino acid sequences, particularly in the putative transmembrane regions and the first and second cytoplasmic loops. This similarity is in marked contrast to the sequence divergence in the amino- and carboxyl-terminal regions and the third cytoplasmic loop. The observed sequence similarity and divergence would thus contribute to the expression of similar but pharmacologically distinguishable activities of the two tachykinin receptors.     

Partial characterization of the androgen receptor of the newborn rat gubernaculum

Androgen receptors were measured by sucrose density gradient techniques in low-salt and high-salt extracts of gubernaculum, urogenital sinus, and bladder from 3- to 5-day-old male rats. Specific 5 alpha-[3H] dihydrotestosterone binding was present in both the low-salt and high-salt extracts of gubernacular tissue. The total amount of receptor in gubernaculum was approximately one-fifth the total amount of androgen binding measured in the urogenital sinus (119 fmol/g tissue in gubernaculum vs. 562 fmol/g tissue in urogenital sinus). No androgen receptor was detected in bladder. A 10-fold excess of nonradioactive 5 alpha-dihydrotestosterone competed well for 5 mM 5 alpha-[3H]-dihydrotestosterone binding, whereas 10-fold molar excesses of testosterone, estradiol, and progesterone were ineffective. These results suggest that the gubernaculum may be a target tissue for androgens.     

Cloning, functional expression and characterization of a human olfactory receptor.

The human olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants begins with the binding of an odorant ligand to a specific receptor protein on the olfactory neuron cell surface. To address the problem of olfactory perception at a molecular level, we have cloned, functionally expressed and characterized the first human olfactory receptor (OR 17-40). Application of a mixture of hundred different odorants elicited a transient increase in intracellular calcium at HEK 293-cells which were transfected with a plasmid containing the receptor encoding DNA and a membrane import sequence. By subdividing the odorant mixture in smaller groups we could identify a single component which represented the only effective substance: helional. Testing some structurally closely related molecules we found only one other compound which also could activate the receptor: heliotropyl acetone. All other compounds tested were completely ineffective. These findings represent the beginning of molecular understanding of odorant recognition in humans.     

Cloning and functional characterization of a human A1 adenosine receptor.

A human brain hippocampus cDNA library was screened by hybridization with a dog A1 adenosine receptor cDNA probe. Sequencing of the resulting clones identified a 978 residue open reading frame encoding a 326 amino acid polypeptide showing 95.7% similarity with the dog A1 adenosine receptor. Individual clones of stably transfected CHO cells expressing the human A1 receptor were obtained and tested for their response to the A1 agonist CPA [N6-cyclopentyladenosine] in the presence of forskolin. One clone was further characterized with respect to membrane binding of various adenosine agonists and antagonists. The rank order of affinities observed was typical of an A1 adenosine receptor. A Kd value of 2.28 nM was determined using [3H]DPCPX [dipropylcyclopentyl-xanthine], an A1 selective antagonist.