Identification of multiple cyclic AMP-binding proteins in developing Dictyostelium discoideum cells

We have purified two cAMP-binding proteins from developing Dictyostelium discoideum cells, which we designate as CABP-1 and CABP-2. Purified CABP-1 consists of two polypeptides of Mr 41,000 and 36,000, which we refer to as CABP-1A and CABP-1B, respectively. Although CABP-1 exhibited specificity for cAMP, it was not labeled at a detectable level when mixed with 8-azidoadenosine 3':5'-monophosphate (8-N3[3H]cAMP). Unlike CABP-1, CABP-2 was labeled efficiently with 8-N3[3H]cAMP. Purified CABP-2 has a molecular weight of 41,000 and an isoelectric point of 5.8-6.0. The physical and biochemical properties of CABP-2 suggest that it is the regulatory subunit of cAMP-dependent protein kinase described by others (de Gunzburg, J., Part, D., Guiso, N., and Veron, M. (1984) Biochemistry 23, 3805-3812; Majerfeld, J. H., Leichtling, B. H., Maligeni, J. A., Spitz, E., and Rickenberg, H. V. (1984) J. Biol. Chem. 259, 654-661). Although CABP-1A and CABP-2 have the same molecular weight, they appear to be encoded by different genes. Two-dimensional gel electrophoresis revealed that the two polypeptides had different isoelectric points. Moreover, monoclonal antibodies raised against CABP-1 did not cross-react with CABP-2. Also, in vitro translation followed by immunoprecipitation showed that these two polypeptides were derived from primary translation products. Our finding of a novel cAMP-binding protein, CABP-1, suggests that cAMP-dependent protein kinase may not be the only intracellular regulator mediating the effects of cAMP in developing D. discoideum cells.     

Search for new cyclic AMP-binding proteins

Today, there is evidence that the cAMP-dependent kinases (PKA) are not the only intracellular receptors involved in intracellular cAMP signalling in eukaryotes. Other cAMP-binding proteins have been recently identified, including some cyclic nucleotide-gated channels and Epac (exchange protein directly activated by cAMP) proteins. All these proteins bind cAMP through conserved cyclic nucleotide monophosphate-binding domains. However, all putative cAMP-binding proteins having such domains, as revealed by computer analysis, do not necessarily bind cAMP, indicating that their presence is not a sufficient criteria to predict cAMP-binding property for a protein.     

Cyclic AMP-binding proteins in human blood platelets detected by photoaffinity labelling

Cyclic AMP inhibits platelet aggregation induced by physiological agents. 8 Azido [³²P]cyclic AMP (N₃ cyclic AMP) has been utilized as a photoaffinity probe to define the cyclic AMP-binding proteins present in unperturbed human platelets and their subcellular fractions. Specificity of cyclic AMP binding was determined by contrasting binding in the presence and absence of excess unlabelled cyclic AMP, cyclic GMP and 5′-AMP. Binding was unaffected by 5′-AMP and obliterated by cyclic AMP. Four major species of binding proteins, 49 000, 42 000, 39 000, 37 000, were obtained in all platelet fractions (crude homeogenate, cytosol, membranes and granules). Two-dimensional gel electrophoresis of platelet cytosol resolved the major molecular weight species into 15 specific cyclic AMP binding proteins of four molecular weight classes differing by charge density. These studies suggest that platelets contain an array of specific cyclic AMP-binding proteins which may function in hemostatic regulation.     

Identification, characterization, and quantitative measurement of cyclic AMP receptor proteins in cytosol of various tissues using a photoaffinity ligand.

Two protein bands, present in cytosol fractions from each of seven rat tissues examined, specifically incorporated 32P-labeled 8-azidoadenosine 3':5'-monophosphate (8-N3-[32P]cAMP), a photoaffinity label for cAMP-binding sites. These proteins had apparent molecular weights of 47,000 and 54,000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. These two proteins were characterized in three of the tissues, namely, heart, uterus, and liver, by the total amount of 8-N3-[32P]cAMP incorporation, by the dissociation constant (Kd) for 8-N3-[32P]cAMP, and by the nucleotide specific inhibition of 8-N3-[32P]cAMP incorporation. Several lines of evidence were obtained that the protein with an apparent molecular weight of 47,000 represents the regulatory subunit of a type I cAMP-dependent protein kinase, while the protein with an apparent molecular weight of 54,000 represents the regulatory subunit of a type II cAMP-dependent protein kinase. Almost all of the cAMP receptor protein found in the cytosol of these tissues, as measured by 8-N3-[32P]cAMP incorporation, was associated with these two protein kinases, in agreement with the idea that most effects of cAMP are mediated through protein kinases. The photoaffinity labeling with 8-N3-[32P]cAMP can be used to estimate quantitatively the amounts of regulatory subunit of type I and type II cAMP-dependent protein kinases in various tissues.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Neurohormonal receptors and cyclic AMP-binding proteins in rabbit tracheal mucosa-submucosa

Neurohormones and drugs that alter in vitro tracheal electrolyte transport and mucus glycoprotein secretion were examined for their ability to alter cyclic nucleotide accumulation in a smooth muscle-free preparation of rabbit tracheal mucosa-submucosa. cAMP levels were increased by beta-adrenergic agonists, histamine, 2-Cl-adenosine and prostaglandin E1. cGMP levels were increased by carbachol. The phosphodiesterase inhibitor isobutylmethylxanthine increased cAMP and cGMP levels and potentiated only the beta-adrenergic effects. The beta-adrenergic effects were blocked by (+/-)-propranolol and the effects of histamine by diphenhydramine, atropine and (+/-)-propranolol. Atropine blocked the carbachol effects. The isolated surface epithelium from rabbit trachea had higher basal cAMP levels and greater response to beta-adrenergic agonists and isobutylmethylxanthine than the mucosa-submucosa. Two major cAMP-binding proteins in the tracheal mucosa-submucosa were identified with the photoaffinity label 8-N3-[32P]cAMP. Agents that increased cAMP levels also decreased photoaffinity labelling, suggesting that these two cAMP-binding proteins were being occupied in the intact cell. The molecular weights of the proteins were 50 000 and 54 000 and correspond in electrophoretic mobility to the regulatory subunits of Type-I and Type-II cAMP-dependent protein kinases, respectively. The results are consistent with the hypothesis that epithelial functions in the airways are modulated by a number of agonists which increase cyclic nucleotide levels. The effects of beta-adrenergic agonists is apparently mediated by activation of adenylate cyclase and subsequent activation of cAMP-dependent protein kinases.     

Photoaffinity labeling of a microtubule-associated cyclic AMP-binding protein

8-Azido cyclic AMP has been used as a photoaffinity probe to identify cyclic AMP-binding proteins in microtubule preparations. Bovine brain microtubule proteins and rabbit muscle protein kinase were incubated with the photoaffinity ligand in reduced light for 15 min, without additions or with 100-fold excess unlabeled cyclic AMP or 5′-AMP. Samples were then irradiated at 254 nm at a distance of 1 cm for 5 min, in ice. After irradiation aliquots were taken for electrophoresis in one or two dimensions. Polypeptides which bound the photoaffinity label were visualized by autoradiography. The apparent molecular weights of the most prominent 8-azido ³²P-cyclic AMP-binding proteins are in the same range as those of the RII of the muscle enzyme. Following two-dimensional electrophoresis the major microtubule-associated cyclic AMP-binding proteins resolve as two spots with about the same pI (~pH 5.0) but slightly different molecular weights. Both spots are in the molecular weight range of the tubulins but they are clearly resolved from the tubulins in the first dimension. Cyclic AMP, but not 5′-AMP blocks the labeling of these proteins. There are low levels of labeling of the tubulins, the high-molecular-weight MAPs and several polypeptides with molecular weights near tubulin but with more basic pI. The photoaffinity probe has demonstrated that the major microtubule-associated cyclic AMP-binding protein of bovine brain is distinct from other RII proteins and from tubulin isomorphs.     

Changing pattern of cyclic AMP-binding proteins during germination of Mucor racemosus sporangiospores.

Interation of cyclic AMP with a profoundly changing pattern of specific binding proteins was shown during aerobic germination of sporangiospores from the fungus Mucor racemosus. 32P-labeled 8-azido-cycli AMP, an analogue of cyclic AMP that forms a covalent linkage with the binding proteins under u.v. light, was used as the ligand. Binding proteins carrying this photoaffinity label were separated by polyacrylamide-gel electrophoresis and identified by radioautography. Equibiltrium dissociation constants (Kd) and binding-response curves in the presence of competing nucleotides were identical for both 8-azido-cyclic [32P]AMP and cyclic [3H]AMP. A quantitative binding assay with both 8-azido-cyclic [32P]AMP and cyclic [3H]AMP over the time course of sporangiospore germination indicated a parallel relationship between cyclic AMP-binding capacity and the intracellular concentrations of cyclic AMP reported in a previous study [Paznokas & Sypherd (1975) J. Bacteriol. 124, 134--139]. Both of these parameters attained transient high values at a time of development when addition of exogenous cyclic AMP prevents hyphal-germ-tube emergence. The measured Kd values did not change during sport germination.     

Identification and partial characterization of laminin binding proteins in immature rat Sertoli cells

Laminin, a major component of basement membrane extracellular matrices, promotes differentiation in a number of cell types, including Sertoli cells. We have identified and characterized Sertoli cells. We have identified and characterized Sertoli cell surface molecules which interact with laminin. Using laminin-Sepharose affinity chromatography and [125I]laminin binding to Sertoli cell plasma membranes, binding proteins have been identified with the Mr 110,000, 67,000, 55,000, 45,000, 36,000, and 25,000. In addition, the Mr 110,000 and 67,000 laminin binding proteins were phosphorylated. The 67,000, 45,000, and 36,000 react with antibodies to the previously characterized laminin receptor and these antibodies stain the basolateral surface of Sertoli cells in vivo. Cultured Sertoli cells stain for laminin receptor both on the cell surface and within the cells. Antiserum to the 32,000 and 67,000 laminin binding proteins partially inhibited spreading of Sertoli cells on a laminin-coated culture dish, suggesting a functional importance of those proteins in Sertoli cell differentiation. The 25,000 and 45,000 laminin binding proteins reacted with integrin antibodies, but no high-molecular-weight forms could be detected. Integrin was localized to the cell surface and intracellularly but antibodies did not block Sertoli cell spreading on laminin. This work represents the first identification and characterization of extracellular matrix binding proteins in an endocrine organ and suggests an important role for the nonintegrin 32/67 laminin binding proteins.     

Identification of cyclic guanosine monophosphate-binding proteins in Drosophila by direct photoaffinity labeling

A procedure for direct photoaffinity labeling with [³²P]cGMP has been used to identify cGMP-binding proteins in Drosophila. This method provides better sensitivity and resolution than previously described direct methods, because the proteins can be visualized by autoradiography following sodium dodecyl sulfate-gel electrophoresis. Labeling is observed with cGMP concentrations as low as 4 × 10^?8m and is specific for cGMP. The sensitivity of the technique is sufficient to permit detection of cGMP-binding proteins in crude extracts. With this technique a single cytoplasmic cGMP-binding protein of subunit M_r 108,000 has been identified in Drosophila embryos and cultured cells.     

Subcellular alterations in cyclic AMP-binding and protein kinase activities during ontogeny in the rat cerebellum

Ontogenic relationships between levels of cyclic AMP-binding activity and protein kinase activity were examined in subcellular fractions of the cerebellum during the first 3 weeks of neonatal life. A progressive increase in cyclic AMP levels was paralleled by an increase in cyclic AMP bindign by the nuclear and cytosol fractions, but not by the mitochondrial or microsomal fractions. Utilization of heat-stable protein kinase inhibitor permtited distinction of the cyclic AMP-dependent from the cyclic AMP-independent form of the protein kinase population. Cyclic AMP-dependent protein kinase increased between days 4 and 20 to represent a progressively greater proportion of the protein kinase population. In all subcellular fractions alterations of cyclic AMP-dependent protein kinase during neonatal development paralleled changes in binding of cyclic AMP to protein in these fractions. In both the nuclear and cytosol fractions cyclic AMP-dependent protein kinase activity increased progressively between days 4 and 20, i.e. 64 ± 6 to 176 ± 16 and 79 ± 12 to 340 ± 12 pmol/min per mg protein, respectively. Cyclic AMP-dependent protein kinase activity in the mitochondrial fraction declined during the postnatal period studied, and in the microsomal fraction it rose to a non-sustained peak at 14 days and fell thereafter. Unlike the cyclic AMP-dependent form, cyclic AMP-independent protein kinase activity did not follow the ontogenetic pattern of cyclic AMP-binding activity. The specific activity of nuclear cyclic AMP-independent protein kinase did not change during days 4–20, and a non-sustained rise of cyclic AMP-independent protein kinase activity in both cytosol and microsomal fractions during the 7th–12th day tended to parallel more closely known patterns of postnatal proliferative growth. The findings reported herein indicate that the ontogenic pattern of cyclic AMP-dependent protein kinase varies between different subcellular fractions of the neonatal cerebellum, that these patterns parallel the changes in cyclic AMP-bidign activity, and suggest that the component parts of the cyclic AMP system may develop as a functional unit.     

Sex pheromone receptor proteins. Visualization using a radiolabeled photoaffinity analog

A tritium-labeled photoaffinity analog of a moth pheromone was used to covalently modify pheromone-selective binding proteins in the antennal sensillum lymph and sensory dendritic membranes of the male silk moth, Antheraea polyphemus. This analog, (E,Z)-6,11-[3H]hexadecadienyl diazoacetate, allowed visualization of a 15-kilodalton soluble protein and a 69-kilodalton membrane protein in fluorescence autoradiograms of electrophoretically separated antennal proteins. Covalent modification of these proteins was specifically reduced when incubation and UV irradiation were conducted in the presence of excess unlabeled pheromone, (E,Z)-6,11-hexadecadienyl acetate. These experiments constitute the first direct evidence for a membrane protein of a chemosensory neuron interacting in a specific fashion with a biologically relevant odorant.