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Using a combination of protein kinase A type II overlay screening, rapid amplification of cDNA ends, and database searches, a contig of 9923 bp was assembled and characterized in which the open reading frame encoded a 1901-amino-acid A-kinase-anchoring protein (AKAP) with an apparent SDS-PAGE mobility of 220 kDa, named human AKAP220 (hAKAP220). The hAKAP220 amino acid sequence revealed high similarity to rat AKAP220 in the 1167 C-terminal residues, but contained 727 residues in the N-terminus not present in the reported rat AKAP220 sequence. The hAKAP220 mRNA was expressed at high levels in human testis and in isolated human pachytene spermatocytes and round spermatids. The hAKAP220 protein was present in human male germ cells and mature sperm. Immunofluorescent labeling with specific antibodies indicated that hAKAP220 was localized in the cytoplasm of premeiotic pachytene spermatocytes and in the centrosome of developing postmeiotic germ cells, while a midpiece/centrosome localization was found in elongating spermatocytes and mature sperm. The hAKAP220 protein together with a fraction of PKA types I and II and protein phosphatase I was resistant to detergent extraction of sperm tails, suggesting an association with cytoskeletal structures. In contrast, S-AKAP84/D-AKAP1, which is also present in the midpiece, was extracted under the same conditions. Anti-hAKAP220 antisera coimmunoprecipitated both type I and type II regulatory subunits of PKA in human testis lysates, indicating that hAKAP220 interacts with both classes of R subunits, either through separate or through a common binding motif(s)., (Copyright 2000 Academic Press.)

The type II cGMP-dependent protein kinase is an enzyme originally isolated from the small intestine, and is thought to be involved in the regulation of intestinal ion transport and fluid secretion. A complementary DNA clone encoding a part of the human type II cGMP-dependent protein kinase was isolated from a cerebellum library. Based on sequence information from this complementary DNA, the 5'-end of the type II cGMP-dependent protein kinase was amplified from human brain messenger RNA using polymerase chain reaction. The composite complementary DNA encoded a 762 amino acid protein with a calculated molecular mass of 87.4 kDa. Messenger RNAs encoding the type II cGMP-dependent protein kinase were detected in small intestine, colon and prostate. By using polymerase chain reaction and Southern blotting on somatic cell hybrids, the gene encoding, the type II cGMP-dependent protein kinase was mapped to human chromosome 4q13.1-q21.1.

The RI alpha mRNA level is induced 3-5 times by FSH or cAMP analogs in primary cultures of rat Sertoli cells. In rat tissues, the RI alpha gene gives rise to three different mRNAs of different size: 3.2, 2.9 and 1.7 kb. In the present study we report that the 1.7 kb transcript has a shorter half-life than the two other mRNAs. In cells which had been pre-stimulated with a cAMP analog, inhibition of transcription stabilizes the two larger, but not the smaller sized RI alpha mRNA. However, in contrast, inhibition of protein synthesis stabilizes all the RI alpha mRNAs. Thus, degradation of various mRNAs coding for the same protein reveals different dependencies on transcription and translation.

Messenger RNA for RII beta is transiently induced (greater than 50-fold) by cAMP analogs in primary cultures of rat Sertoli cells. The induction is dependent on protein synthesis. We have previously shown that mRNA for RII beta is stabilized by cAMP, as well as inhibitors of transcription and translation. This indicated that rapid degradation of RII beta mRNA involved a protein with a rapid turnover and its corresponding mRNA. The two RNA synthesis inhibitors used in the present study stabilized both nuclear and cytoplasmic RII beta mRNA, whereas inhibition of protein synthesis stabilized RII beta mRNA in the cytoplasm only. These results indicate that only cytoplasmic degradation of RII beta mRNA is dependent on a protein with high turnover. In contrast, nuclear degradation appears to be dependent on an RNA with a short half-life, not involving protein synthesis.

Northern analysis shows that Go alpha mRNA is highly expressed in immature germ cells from rat. Whereas alpha o2 mRNA is the major form in pachytene spermatocytes, a message of shorter chain length is present in large amount in haploid germ cells. This mRNA was detected with an oligonucleotide specific for the 3'-coding region of alpha o2, but did not hybridize to oligonucleotides specific for the 5'-untranslated and 5'-coding regions. The results indicate the presence of a novel splice variant of alpha o mRNA, which may code for a Go alpha protein important for germ cell development.

We have examined the effect of the protein kinase C activator, TPA, on mRNA levels for subunits of cAMP-dependent protein kinases in the human colonic cancer cell line HT-29, subline m2. Messenger RNA for the regulatory subunit, RI alpha, of cAMP-dependent protein kinases was shown to be present and regulated by TPA. Other mRNAs for subunits of cAMP-dependent protein kinases (RI beta, RII alpha, RII beta, C alpha, C beta) were also present in these cells, but revealed no or only minor changes upon TPA stimulation. When HT-29 cells were cultured in the presence of 10 nM TPA for various time periods, a biphasic response was observed in RI alpha mRNA levels with a maximal increase (approximately 4 fold) after 24 hours. TPA stimulated RI alpha mRNA increased in a concentration-dependent manner and maximal response (4-8 fold) was seen at 3-10 nM. The TPA-induced increase in RI alpha mRNA was not obtained when cells were incubated with TPA together with the protein kinase C inhibitors, staurosporine or H7. The cAMP-analog 8-CPTcAMP alone induced RI alpha mRNA levels 50% more than TPA. Combined treatment with TPA (10 nM) and 8-CPTcAMP (0.1 mM) gave an increase in RI alpha mRNA similar to TPA. These results demonstrate an interaction between the protein kinase C pathway and mRNA levels for the RI alpha subunit of cAMP-dependent protein kinases in HT-29 cells.

The 1.9 kb mRNA encoding the alpha subunit of Gs observed in the somatic cells of the rat testis was not seen in the germ cells (pachytene spermatocytes and round spermatids) as revealed by Northern blot analysis. Interestingly, the cDNA probe for Gs alpha detected a mRNA of shorter chain length (0.9 kb) exclusively in the haploid germ cells (round and elongated spermatids). However, no ADP ribosylated protein was detected in membranes from germ cells after cholera toxin treatment. The 0.9 kb transcript may represent a haploid germ cell-specific variant of Gs alpha, which is functionally different from the stimulatory component of the adenylate cyclase system.

The regulatory subunit of cAMP-dependent protein kinase designated RII beta (RII51) has previously been shown to be the product of a separate gene. This was accomplished by the molecular cloning of a partial cDNA clone estimated to lack 30-45 nucleotides of the 5' end of the coding region. We hereby report the isolation of a cDNA clone for RII beta from rat granulosa cells, extending 43 nucleotides further 5' compared with the previously published cDNA sequence, and from which the entire amino acid sequence (415 residues) of the rat RII beta protein can be deduced. A cAMP regulated mRNA of 3.2 kilobases (kb) for RII beta was detected by the isolated cDNA in rat Sertoli cells.

A 1.5 kilobase (kb) cDNA clone containing the entire coding region for a regulatory subunit of type I cAMP-dependent protein kinase (RI) was isolated from a human testis cDNA library. The cDNA clone encodes a protein of 381 amino acids that shows 98% and 97% homology to the bovine skeletal muscle RI and rat brain RI, respectively. Northern blot analysis demonstrates two major mRNA-species (1.5 and 3.0 kb) in human testis and one mRNA-species (3.0 kb) in human T-lymphocytes.

This report describes a simple procedure to quantitate receptors for 1,25-dihydroxyvitamin D3 in the presence of contaminating serum vitamin D binding protein. The method ("differential displacement") takes advantage of the greatly different rates of dissociation of 1,25-dihydroxyvitamin D3 from the serum vitamin D binding protein (t 1/2 less than 5 min) and from the receptor (t 1/2 greater than 120 h) at 0 degrees C. The principle of "differential displacement" can be used for other steroid receptors as well, and in combination with a variety of different binding assays, provided they are performed under conditions where the dissociation of the steroid from the receptor is slow.

The pattern of androgenic metabolites in blood, muscle, caput and cauda epididymidis has been investigated in functionally hepatectomized 24 hours castrated rats, 3 hours after the intra-muscular injection of 200 muCi of 3H-3H-3alpha-diol. Identification of the radioactive metabolites showed only negligible differences between the epididymal regions. In both caput and cauda the main metabolite was DHT (17beta-hydroxy-5alpha-androstane-3-one); 3alpha- and 3beta-diol, androsterone (3alpha-hydroxy-5alpha-androstane-17-one), 5-A-dione (5alpha-androsterone-3, 17-dione), delta16-3alpha-01 (kalpha-androst-16-en-3alpha-01), delta16-3beta-01 (5alpha-androst-16-en-3alpha-01) and delta16-3-one (5alpha-androst-16-en-3-one) were also present. Androsterone and 3alpha-diol were the predominant metabolites in blood and muscle. No delta16 compounds could be detected and in constrast to epididymis, more than 50% of the radioactivity was associated with polar compounds. From determination of total radioactivity, it was seen that retention by epididymis varied from two to four times that of muscle. Purification and identification of the radioactivity associated with the nuclear fraction demonstrated that DHT was the only nuclear bound androgen. It is suggested from these results that at least one effect of 3alpha-diol on the rat epididymis is exerted through its conversion to DHT.

The levels of mRNA for cellular retinol binding protein (CRBP) were studied in primary rat Sertoli cell cultures treated with cAMP analogues and retinol. In the presence of cyclic AMP analogues a dose- and time-dependent reduction (70-90%) of the levels of mRNA for CRBP was observed. Retinol concentrations above 10 nM induced a dose- and time-dependent increase (2-3 fold) in mRNA levels for CRBP. Assuming that CRBP is important for vitamin A action, our data indicate that both cAMP and retinol itself modulate the sensitivity of the Sertoli cells for retinol.

Testis nuclei of hypophysectomized rats selectively accumulate labeled testosterone and 5alpha-dihydrotestosterone following the injection of tritiated testosterone in vivo. Testosterone and 5alpha-dihydrotestosterone are bound to macromolecules in nuclei and can be extracted with 0.5 M KCl. Accumulation of protein bound radioactive androgens in nuclei of isolated seminiferous tubules is similar to that of whole testis. The relative amounts of testosterone and dihydrotestosterone in purified nuclei were similar to the relative amounts bound to cytoplasmic receptors, suggesting that cytoplasmic androgen-receptor complexes may be transported into the nuclei. Binding of labeled androgen is saturable and inhibited by prior injection of unlabeled testosterone or cyproterone acetate. Nuclear binding sites are destroyed by the proteolytic enzyme pronase, but not by DNase. Like the cytoplasmic androgen-receptor complexes in rat testis, nuclear androgen-protein complexes are heat labile and dissociate slowly at 0 degrees C. androgens fail to accumulate in testis nuclei of the Stanley-Gumbreck androgen insensitive rat, a species lacking cytoplasmic androgen receptors in testis and other androgen target tissues.