Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa.

BACKGROUND: There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. METHODS: Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of (13)C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. RESULTS: The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed (13)C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. CONCLUSIONS: Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD(+) through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

Identification of novel splice variants of the human catalytic subunit Cbeta of cAMP-dependent protein kinase.

Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.

HA95 is a protein of the chromatin and nuclear matrix regulating nuclear envelope dynamics.

We report a role for HA95, a nuclear protein with high homology to the nuclear A-kinase anchoring protein AKAP95, in the regulation of nuclear envelope-chromatin interactions. Biochemical and photobleaching data indicate that HA95 is tightly associated with chromatin and the nuclear matrix/lamina network in interphase, and bound to chromatin at mitosis. HA95 resides in a complex together with lamin B receptor (LBR), lamina-associated polypeptide (LAP)2 and emerin, integral proteins of the inner nuclear membrane. Cross-linking experiments, however, illustrate a tight association of HA95 with LBR and LAP2 only. Intra-nuclear blocking of HA95 with anti-HA95 antibodies abolishes nuclear breakdown in a mitotic HeLa cell extract. The antibodies inhibit nuclear membrane breakdown and chromatin condensation - the latter independently of nuclear membranes. However, lamina disassembly is not affected, as judged by immunological analyses of A/C- and B-type lamins. In contrast, immunoblocking of HA95 bound to condensed chromosomes does not impair chromatin decondensation, nuclear membrane reassembly or lamina reformation. Our results argue for a role for HA95 in anchoring nuclear membranes and lamins to chromatin in interphase, and in releasing membranes from chromatin at mitosis. The data also suggest that HA95 is not involved in initial binding of membranes to chromatin upon nuclear reassembly. We propose that HA95 is a central platform at the chromatin/nuclear matrix interface implicated in regulating nuclear envelope-chromatin interactions during the cell cycle.

A novel isoform of human cyclic 3',5'-adenosine monophosphate-dependent protein kinase, c alpha-s, localizes to sperm midpiece.

Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human C alpha catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine C alpha gene. Sequence comparison revealed similarity to an ovine C alpha variant characterized by protein purification and micropeptide sequencing, C alpha-s, identifying the cloned human cDNA as the C alpha-s isoform. The C alpha-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene spermatocytes was demonstrated. The C alpha-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a C alpha-s-specific antibody indicated that C alpha-s was localized in the midpiece region of the spermatozoon. The majority of C alpha-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the C alpha-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms RI alpha, RII alpha, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target C alpha-s. This novel C alpha-s splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.

Localization of a novel human A-kinase-anchoring protein, hAKAP220, during spermatogenesis.

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).

Identification, cloning and characterization of a novel nuclear protein, HA95, homologous to A-kinase anchoring protein 95.

Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N-terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c-myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co-localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co-localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.

Identification and quantitation of cAMP-dependent protein kinase R subunit isoforms in subcellular fractions of failing human myocardium.

Isoforms of regulatory (R) subunits of cAMP-dependent protein kinase were identified immunochemically and quantified in soluble and washed particulate fractions of failing human left ventricular myocardium. The predominant isoforms in both fractions were RI alpha and RII alpha. Both isoforms were present in comparable amounts in these fractions, although RII alpha subunits were somewhat more prevalent than RI alpha subunits in washed particulate fractions. The ratio of R subunits to catalytic (C) subunits was three-fold higher in soluble than in particulate fractions. Identical observations were made in preparations from non-failing human left ventricular myocardium. Since RI and RII have different affinities for cAMP and may direct catalytic activity to different substrates, the presence of both subunits in both soluble and particulate fractions provides a mechanism whereby the compartment-selective changes in cAMP content that have been described in failing human myocardium may affect not only the level but also the profile of protein phosphorylation in these compartments. The high R:C subunit ratio in soluble fractions suggests that cytosolic kinase activity in human myocardium may be less sensitive to changes in cAMP content than membrane-bound kinase activity, and this may contribute to the different effects of increases in soluble and particulate cAMP content on intracellular Ca2+transients and contraction and relaxation.

Differential localization of protein kinase A type II isozymes in the Golgi-centrosomal area.

Selectivity in the action of cAMP may be mediated by compartmentalized pools of cyclic AMP-dependent protein kinase (PKA). PKA type II is directed to different subcellular loci by interaction of the type II regulatory subunits (RIIalpha, RIIbeta) with A-kinase anchoring proteins. In order to separately investigate the subcellular localization of PKA type II isozymes, monospecific antibodies to human RIIalpha and RIIbeta subunits of PKA were developed. We demonstrate that centrosomes bind both RIIalpha and RIIbeta. Centrosomes were the preferred intracellular anchoring site for RIIbeta. However, centrosomal localization of RIIbeta was observed only in some highly differentiated cells such as keratinocytes, granulosa cells, and macrophages and in all neoplastic cell lines examined. Centrosomal localization of RIIbeta was not observed in normal undifferentiated cells such as fibroblasts, myoblasts, and T and B cells. In contrast, RIIalpha was abundant in the Golgi area and in the trans-Golgi network (TGN). Furthermore, although RIIalpha appeared to colocalize with microtubules in the Golgi/TGN, extractions with nonionic detergent demonstrated that RIIalpha was mainly membrane-associated. In addition, alterations of microtubule dynamics with Nocodazole or Taxol affected the distribution of the detergent-extractable pool of RIIalpha, indicating that RIIalpha may localize with microtubule-associated vesicles. Thus, RIIalpha and RIIbeta clearly localize differently in the Golgi-centrosomal region. This indicates specific roles for PKA isozymes containing either RIIalpha or RIIbeta.

Cloning and characterization of a cDNA encoding an A-kinase anchoring protein located in the centrosome, AKAP450.

A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A-kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21-22. This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was designated AKAP450 (DDBJ/EMBL/GenBank accession No. AJ131693). Sequence comparison demonstrated that the open reading frame contained a previously characterized cDNA encoding Yotiao, as well as the human homologue of AKAP120. Numerous coiled-coil structures were predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins was observed. A putative RII-binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA-buffer extracts of HeLa cells demonstrated co-precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it was demonstrated that AKAP450 localized to centrosomes. Furthermore, AKAP450 was shown to co-purify in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene.

Mapping of the gene encoding the regulatory subunit RII alpha of cAMP-dependent protein kinase (locus PRKAR2A) to human chromosome region 3p21.3-p21.2.

We have determined the chromosomal localization of the gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase (locus PRKAR2A) to human chromosome 3 using polymerase chain reaction (PCR) and Southern blot analysis of two different somatic cell hybrid mapping panels. Furthermore, PCR analysis of a chromosome 3 mapping panel revealed the presence of a human RII alpha-specific amplification product only in cell lines containing the region 3p21.3-p21.2. The localization of PRKAR2A was confirmed by PCR mapping using the Stanford G3 Radiation Hybrid Panel as template. The results from this analysis demonstrated that PRKAR2A is most closely linked to D3S3334 (lod score 12.5) and flanked by D3S1322E and D3S1581.

Characterization of the gene encoding the human type II cGMP-dependent protein kinase.

The type II cGMP-dependent protein kinase (cGK) plays a pivotal role in the regulation of intestinal fluid balance in man. Furthermore, mice carrying a null mutation for the gene encoding the type II cGK develop as dwarfs indicating that this enzyme has other less characterized roles. The present report describes the isolation and characterization of bacterial artificial chromosome (BAC)- and P1-derived artificial chromosome (PAC)-clones containing the gene encoding the human type II cGK. The gene was estimated to cover at least 125 kb and consisted of 19 exons separated by introns of various lengths. The splice junctions of the type II cGK gene corresponded well with the structure of the gene encoding human type I cGK and with the splice junctions observed in the Drosophila melanogaster DG2 gene. 5'-rapid amplification of cDNA-ends established the presence of a non-translated exon.

The gene encoding the C gamma catalytic subunit of cAMP-dependent protein kinase is a transcribed retroposon.

Three different catalytic isoforms of cAMP-dependent protein kinase have been identified (C alpha, C beta, and C gamma). We report the cloning and characterization of the human and rhesus monkey genes encoding the testis-specific C gamma subunit. The human C gamma gene is intronless with an open reading frame similar to the previously published cDNA sequence. The 3' and 5' flanking regions share high similarity with the C alpha nontranslated regions (82%) also outside the regions corresponding to the C gamma cDNA. The human gene is flanked by an Alu-related sequence in the 5'-end and there are insertions of two Alu-related sequences in the 3' nontranslated region. The observation that the C gamma gene is intronless and colinear with C alpha mRNA, together with the presence of remnants of a poly(A) tail and flanking direct repeats, indicates that the C gamma gene is a C alpha-derived retroposon. The 5' flanking region of this gene has a high G/C content and a putative TATA box situated at -138 compared to the translation initiation codon. Cloning and sequencing of a partial C gamma rhesus monkey gene demonstrate conservation of the sequence in primates. Northern analysis on isolated and fractionated human germ cells of testes from normal and estrogen-treated individuals demonstrates that the C gamma gene is expressed only in germ cells in the human testis. Our results indicate that the C gamma gene is a retroposon specifically transcribed in primate testicular germ cells.

Localization of cAMP-dependent signal transducers in early rat liver carcinogenesis.

Cyclic AMP (cAMP) is an important regulator of liver growth and differentiation. The main intracellular cAMP receptor, cAMP-dependent protein kinase (PKA), consists of two regulatory (R) and two catalytic (C) subunits. There are two classes, RI and RII, of the regulatory subunit, giving rise to type I (RI2C2) and type II (RII2C2) PKA. The RI/RII ratio generally decreases during organ development, and increases during carcinogenesis. Alterations in this ratio have been implicated as an important factor in experimental and clinical carcinogenesis. We have studied the expression of RIalpha, RIIalpha, Calpha, and an important substrate of PKA, the cAMP-response element binding protein, during rat liver carcinogenesis. Two-color immunofluorescence and confocal laser scan microscopy were used to characterize localization of the cAMP-dependent signal transducers in hepatocytes, bile ducts, oval cells, and preneoplastic lesions. We found that bile ducts and oval cells (putative liver stem cells) contained a higher RI/RII ratio than hepatocytes and preneoplastic lesions. Thus, an altered RI/RII ratio was not detected during early rat liver carcinogenesis, but may contribute to differentiation of putative liver stem cells to hepatocytes.

Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95.

The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs). A full-length human clone encoding AKAP95 was identified and sequenced, and revealed a 692-amino acid open reading frame that was 89% homologous to the rat AKAP95 (V. M. Coghlan, L. K. Langeberg, A. Fernandez, N. J. Lamb, and J. D. Scott (1994) J. Biol. Chem. 269, 7658-7665). The gene encoding AKAP95 was mapped to human chromosome 19p13.1-q12 using somatic cell hybrids and PCR. A fragment covering amino acids 414-692 of human AKAP95 was expressed in Escherichia coli and shown to bind RIIalpha. Competition with a peptide covering the RII-binding domain of AKAP Ht31 abolished RIIalpha binding to AKAP95. Immunofluorescence studies in quiescent human Hs-68 fibroblasts showed a nuclear localization of AKAP95, whereas RIIalpha was excluded from the nucleus. In contrast, during mitosis AKAP95 staining was markedly changed and appeared to be excluded from the condensed chromatin and localized outside the metaphase plate. Furthermore, the subcellular localizations of AKAP95 and RIIalpha overlapped in metaphase but started to segregate in anaphase and were again separated as AKAP95 reentered the nucleus in telophase. Finally, RIIalpha was coimmunoprecipitated with AKAP95 from HeLa cells arrested in mitosis, but not from interphase HeLa cells, demonstrating a physical association between these two molecules during mitosis. The results show a distinct redistribution of AKAP95 during mitosis, suggesting that the interaction between AKAP95 and RIIalpha may be cell cycle-dependent.

Characterization of the human gene encoding the type I alpha and type I beta cGMP-dependent protein kinase (PRKG1).

The type I cGMP-dependent protein kinase (cGK) has been shown to play a crucial role in the relaxation of vascular smooth muscle by lowering the intracellular level of calcium. Two isoforms of type I cGK have been described, type I alpha and type I beta, differing only in their N-terminal parts. This report describes the cloning of the gene PRKG1 encoding both human type I cGK isoforms. PRKG1 is a single-copy gene consisting of 19 exons encompassing at least 220 kb. Several of the splice sites previously observed in the Drosophila melanogaster DG2 gene have been conserved in PRKG1, and these conserved splice sites correlated well with the boundaries between several of the previously proposed functional domains of type I cGK. The first two exons of the type I cGK gene were shown to encode the type I alpha- and type I beta-specific parts of the cGK. Using 5'-rapid amplification of cDNA ends, potential sites for transcription initiation were identified 5' upstream of both these exons. Northern blot analyses demonstrated distinct patterns of expression of the isoforms of type I alpha and I beta cGK in different human tissues.

Characterization of the 5'-flanking region of the gene for the cAMP-inducible protein kinase A subunit, RIIbeta, in Sertoli cells.

Activation of cyclic AMP-dependent protein kinases (protein kinase A, PKA) by gonadotropins and cyclic AMP (cAMP) plays an important role in the regulation of testicular functions. A regulatory subunit, RIIbeta, of PKA is transcriptionally induced in rat Sertoli cells in response to treatment with cAMP. The present study addresses regulatory mechanisms leading to increased transcription of the rat RIIbeta gene. We have localized a footprint which overlaps one of the major transcription initiation sites in the basal promoter (-293 to -123). One of the proteins binding this sequence belongs to the NF-1 family of transcription factors. We also observed binding to a basic helix-loop-helix (bHLH) response element. Furthermore, transfection studies of various 5'-deletions of the rat RIIbeta gene in primary cultures of rat Sertoli cells and in peritubular cells revealed the presence of an upstream region (-723 to -395, cAMP-responsive region) inhibiting basal expression from the rat RIIbeta gene only in Sertoli cells. This region was found to enhance cAMP responsiveness in Sertoli cells but not in peritubular cells. Interactions with downstream elements seemed to be important for the function of the cAMP-responsive region. Although some short stretches reveal homology to the cAMP-responsive regions of other slowly cAMP-responding genes, and an AP-1-like element is present, no strong resemblance to any known regulatory element responsive to cAMP is found.

Structure, function, and regulation of human cAMP-dependent protein kinases.

A large number of hormones, neurotransmitters, and other signaling substances that bind to G-protein-coupled cell-surface receptors have their signals converge at one sole second messenger, cAMP. The question of how specificity can be maintained in a signal-transduction system in which many extracellular signals leading to a vast array of intracellular responses are all mediated through one second-messenger system has been the subject of thorough investigation and a great deal of speculation. An increasing number of cAK isozymes, consisting of homo- or heterodimers of R subunits (RIalpha, RIbeta, RIIalpha, RIIbeta) with associated catalytic subunits (C alpha, Cbeta, Cgamma), may, at least in part, explain this specificity. The various cAK isozymes display distinct biochemical properties, and the heterogeneous subunits of cAK reveal cell-specific expression and differential regulation at the level of gene transcription, mRNA stability, and protein stability in response to a wide range of hormones and other signaling substances. The existence of a number of anchoring proteins specific to either RIIalpha or RIIbeta, and which localize cAKII isozymes toward distinct substrates at defined subcellular loci, strongly supports the idea that specific functions can be assigned to the various cAK isozymes. The demonstration that selective activation of cAKI is necessary and sufficient for cAMP-mediated inhibition of T-cell proliferation, and the observation that T-cell activation is associated with redistribution and colocalization of cAKI to the TCR, is also compatible with the notion of isozyme-specific effects.

Molecular cloning, upstream sequence and promoter studies of the human gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase.

The gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase is highly regulated during spermatogenesis and a strong signal from a distinct short mRNA form is observed postmeiotically during spermatid elongation. This report presents the isolation and characterization of the 5'-flanking region (1.2 kb) and exon 1 of the human RII alpha gene. S1 nuclease mapping and primer extension experiments revealed the presence of a major transcriptional start site located 208 nucleotides upstream of start for translation. The 5'-flanking region of the RII alpha gene did not contain a TATA box and was highly G/C-rich. A basal promoter directing high levels of chloramphenicol acetyl transferase (CAT) activity was identified in the 5'-flanking sequence. Several potential binding sites for transcription factors were identified in this region, which may be responsible for the germ cell-specific regulation of this gene. We have previously reported that the human testis RII alpha cDNA contains a region (amino acids 45-75) with little or no homology to the corresponding rat skeletal muscle cDNA (Oyen, O., Myklebust, F., Scott, J.D., Cadd, G.G., McKnight, G.S., Hansson, V. and Jahnsen, T. (1990) Biol. Reprod. 43, 46-54). We examined whether this difference could arise due to organ-specific splice mechanisms or represented a species difference. We show that the low homology region of the human RII alpha cDNA resides entirely within exon 1, and does not originate from a tissue-specific alternate splicing of this distinct region.

The human gene for the regulatory subunit RI alpha of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase: two distinct promoters provide differential regulation of alternately spliced messenger ribonucleic acids.

The present study reports the exon-intron organization of the human RI alpha gene of cAMP-dependent protein kinase and approximately kilobases (kb) of the 5'-flanking region obtained by isolation and sequencing of several phage clones from human genomic libraries. The RI alpha gene is composed of nine coding exons of varying lengths, separated by introns, giving the gene a total length of at least 21 kb. our recent cloning of a processed RI alpha pseudogene with a 5'-noncoding region different from the previously reported RI alpha complementary RNA indicated that the RI alpha gene may have multiple leader exons giving rise to alternately spliced messenger RNAs (mRNAs). Reverse transcription of human testis RNA followed by PCR identified two different RI alpha mRNA species (RI alpha 1a and RI alpha 1b) containing distinct sequences due to alternately splicing the gene. The previously known RI alpha 1b mRNA revealed low constitutive expression in a human B lymphoid cell line (Reh) and was stimulated only 4- to 6-fold by treatment with cAMP. In contrast, very low levels of the novel RI alpha 1a mRNA were present in untreated Reh cells, but were stimulated 40-to 50-fold by cAMP. The 5'-flanking sequence of the RI alpha gene was G/C rich and did not contain any TATA box. Several putative transcription initiation sites were identified in front of each leader exon (exons 1a and 1b) by the 5'-rapid amplification of complementary DNA ends technique. To determine whether the sequences 5' of both leader exons had promoter activities, the 5'-flanking sequences of exons 1a and 1b were inserted in front of a chloramphenicol acetyltransferase reporter gene, and their ability to direct transcription were examined. Transfection of these constructs into rat GH4C1 cells demonstrated that both constructs had promoter activities, as evidenced by high levels of chloramphenicol acetyltransferase activity.