Cloning of the human serotonin 5-HT2 and 5-HT1C receptor subtypes.

We report the cloning and the deduced amino acid sequence of cDNAs encoding both the human serotonin 5-HT2 and 5-HT1C receptors. The human 5-HT2 and 5-HT1C receptors shared 87% and 90% amino acid homology, respectively, with their rat counterparts. The most divergent regions of the 5-HT2 receptor between human and rat were the N-terminal extracellular domain (75% homology) and the C-terminal intracellular domain (67% homology between amino acids 426-474). The greatest variability between the human and rat 5-HT1C receptors were at the N-terminal extracellular domain (78% homology) and the third cytoplasmic loop (71% homology). The availability of the cloned human 5-HT2 and 5-HT1C receptors will help facilitate the further understanding of the molecular pharmacology and physiology of these receptors.

Promoter specificity and modulation of RNA polymerase II transcription.

RNA polymerase II is a multisubunit enzyme involved in the transcription of protein encoding genes. Recently acquired knowledge of the transcription process and of the RNA polymerase molecule as well as the isolation of subunit clones have led to a better understanding of the enzyme's functional regulation. Specific transcription initiation occurs at promoter regions located upstream of the gene and requires a minimum of five basic factors in addition to the enzyme. Furthermore, proteins that bind to specific DNA elements within the promoter also regulate transcriptional activity. Additional factors are required for the elongation and, possibly, termination of transcription. Two elongation factors, SII and TFIIF, interact directly with the RNA polymerase II molecule. Functional domains of RNA polymerase II have been determined by analysis of genomic clones for the two largest subunits of the enzyme. For example, the 240-kDa largest subunit contains a highly phosphorylated carboxyl-terminal heptapeptide domain repeated 26-52 times that is absolutely required for transcription in vivo. Analysis of the polymerase molecule and its interaction with basic gene-specific transcription factors will aid in our studies of the control of gene expression.

Identification of glutathione S-transferase Yb1 mRNA as the androgen-repressed mRNA by cDNA cloning and sequence analysis.

Androgens, while stimulating the growth of the rat ventral prostate, can also repress the levels of a limited number of mRNAs. The cDNA for one of the androgen-repressed mRNAs has been identified by nucleotide sequence analysis as coding for the glutathione S-transferase Yb1 subunit. The prostate cDNA is 1071 nucleotides long, and only 2 or 4 bases of this sequence do not match the two published sequences of the cDNA for the Yb1 subunit of rat liver glutathione S-transferase. The amino acids in the protein encoded by the prostate cDNA matched completely with that for one of the liver cDNAs and differ with the other cDNA only in two of 218 amino acids. The identification of the androgen-repressed mRNA as a glutathione S-transferase subunit may indicate that some of the cellular actions of the enzyme may be important in the control of androgen-dependent growth of the prostate. Since Yb forms of the transferases have been colocalized with uridylic acid-rich small nuclear RNAs at interchromatinic regions of the cell nucleus, autoregulation of prostate growth by androgens may be carried out through the modulation of RNA production or processing in this target organ.

Prostatic spermine-binding protein. Cloning and nucleotide sequence of cDNA, amino acid sequence, and androgenic control of mRNA level.

cDNA for mRNA of an androgen-dependent spermine-binding protein (SBP) of rat ventral prostate was cloned by inserting cDNA into a dG-tailed expression vector, pUC8, and screening the expression library with anti-SBP antibodies. Hybrid-selected translation using plasmid DNA from positive clones yielded a 34-kDa protein which was immunoprecipitated by affinity-purified anti-SBP antibodies. SBP mRNA is about 1260 bases long as measured by Northern blot hybridization. An amino acid sequence deduced from the nucleotide sequence of the cDNA was identical to an amino acid sequence found in SBP. SBP is extremely rich in acidic residues. Aspartic and glutamic acids, which make up about 33% of the total sequence, comprise 89 of a stretch of 126 amino acids at the carboxyl-terminal end. By dot hybridization analysis, SBP mRNA was not detected in rat liver, kidney, brain, submaxillary gland, or uterus. The prostate levels of SBP mRNA were measured by mRNA translation and dot hybridization. SBP mRNA level decreased to less than 20% of normal 2 days after castration of rats, and this decrease was reversed by 5 alpha-dihydrotestosterone injection into castrated rats.

Androgen repression of the production of a 29-kilodalton protein and its mRNA in the rat ventral prostate.

The regression of the ventral prostate, after a rat is deprived of androgens by castration, is accompanied by a marked decrease in the prostate's ability to synthesize RNA and major proteins. Surprisingly, in vitro translation of prostate RNA, isolated from rats 2 days after castration, detects four proteins with Mr of approximately 29,000, 37,000, 46,000, and 49,000 whose message levels increased 4- to 12-fold compared to results from normal rats. According to cDNA dot hybridization analysis, the increase after castration in the level of the 29-kDa protein-mRNA (per unit amount of DNA) was reversed within 6 h by androgen treatment of castrated rats. In contrast, the level of a mRNA in male rat liver, which hybridized to a cloned probe for the prostate 29-kDa protein-mRNA was reduced by castration and increased by androgen treatment. During an in vitro incubation, the ventral prostates of normal rats were much less efficient than the prostates of rats castrated 2 days earlier in synthesizing a 29-kDa protein. Despite the fact that androgenic manipulation of rats induced very rapid and significant changes in the production of the 29-kDa protein and in the level of its mRNA, the cellular level of this protein in the prostate, as determined by radioimmunoassay, was maintained at near normal values throughout the 2-week experimental period. Thus, the prostate appears to have a mechanism, based on androgen repression of certain genes, to maintain the cellular levels of the 29-kDa protein and possibly other structurally or functionally important proteins during both the periods of androgen-dependent growth and the castration-induced regression. The loss of such a regulatory mechanism may result in androgen-independent abnormal prostate growth.