Rameshwar K. Sharma

Dual regulation of atrial natriuretic factor-dependent guanylate cyclase activity by ATP

The ‘second messenger’ of certain atrial natriuretic factor (ANF) signals is cyclic GMP. One type of ANF receptor linked to the synthesis of cyclic GMP is a transmembrane protein which contains both the ANF‐binding and guanylate cyclase activities. The consensus is that the maximal activity or this guanylate cyclase is observed in the presence of ATP. We now show that depending upon the cofactors Mg2+ or Mn2+, ATP stimulates or inhibits the ANF‐dependent guanylate cyclase activity in the testicular plasma membranes; stimulation in the presence of Mg2+ and inhibition in the presence of Mn2+. With Mg2+ as cofactor neither ATP nor ANF stimulate the cyclase activity — it is only when the two are together that the enzyme is activated. Furthermore, this investigation for the first time demonstrates binding of ATP to the ANF receptor guanylate cyclase, suggesting that ATP‐mediated responses could occur by direct ATP binding to the cyclase.

Molecular cloning, sequencing and expression of an α2-adrenergic receptor complementary DNA from rat brain

We have isolated a cDNA clone from rat brain using a human platelet α2-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney α2-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3′-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5′-untranslated region. Southern-blot analysis with a human platelet α2-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain α2-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A+) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the α2-adrenergic antagonist [3H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the α2A subclass. We conclude that the rat brain cDNA encodes a new α2-adrenergic receptor subtype that may be brain-specific.

Characterization and regulation by protein kinase C of renal glomerular atrial natriuretic peptide receptor-coupled guanylate cyclase

The nature and regulation of atrial natriuretic peptide (ANP)-sensitive guanylate cyclase in rat renal glomerular membranes was examined. By affinity crosslinking techniques, three bands with apparent molecular masses of 180, 130 and 64 kDa were specifically labeled with [125I]ANP. A specific antibody to the 180 kDa membrane guanylate cyclase of rat adrenocortical carcinoma recognized a 180 kDa band on Western blot analysis of solubilized, GTP-affinity purified glomerular membrane proteins. The same antibody completely inhibited ANP-stimulated guanylate cyclase activity in glomerular membrane fractions. Partially purified protein kinase C inhibited ANP-stimulated guanylate cyclase activity in glomerular membrane fractions. It is concluded that a 180 kDa ANP-sensitive guanylate cyclase is present in glomerular membranes, and that this enzyme is inhibited directly by protein kinase C.

Regulation of guanylate cyclase activity by atrial natriuretic factor and protein kinase C.

SummaryThe putative ‘second messenger’ of certain atrial natriuretic factor (ANF) signal transductions is cyclic GMP. Recently, we purified a 180-kDa protein, apparently containing both ANF receptor and guanylate cyclase activities, and hypothesized that this is one of the cyclic GMP transmembrane signal transducers. The enzyme is ubiquitous and appears to be conserved. Utilizing the 180-kDa membrane guanylate cyclase, we now show that the 180-kDa guanylate cyclase is regulated in opposing fashions by two receptor signals—ANF stimulating it and protein kinase C inhibiting it. Furthermore, protein kinase C phosphorylates the 180-kDa enzyme. This suggests a novel ‘switch on’ and ‘switch off’ mechanism of the cyclic GMP signal transduction. ‘Switch off’ represents the phosphorylation while ‘switch on’ the dephosphorylation of the enzyme.

Genetically tailored atrial natriuretic factor‐dependent guanylate cyclase Immunological and functional identity with 180 kDa membrane guanylate cyclase and ATP signaling site

Biochemical and immunological studies have established that one of the signal transducers of atrial natriuretic factor (ANF) is a 180 kDa membrane guanylate cyclase (180 kDa mGC), which is also an ANF receptor; obligatory in the transduction process is an intervening ATP‐regulated step, but its mechanism is not known. GCα is a newly discovered member of the guanylate cyclase family whose activity is independent of the known natriuretic peptides, and the enzyme is not an ANF receptor. The genetically tailored GCα, GCα‐DmutGln338 Leu364, however, is not only a guanylate cyclase but also an ANF receptor and is structurally and functionally identical to the cloned wild‐type ANF receptor guanylate cyclase, GC‐A. We now report that the ANF‐dependent guanylate cyclase activity in the particulate fractions of cells transfected with GCα‐DmutGln338 Leu364 was inhibited by the 180 kDa mGC polyclonal antibody, and with this antibody probe it was possible to purify the 130 kDa expressed receptor; the hormone‐dependent cyclase activity of this receptor was exclusively dependent upon ATP; and through site‐directed mutational studies with GCα mutants, the signaling sequence that defines ATP binding site was identified. We thus conclude that 180 kDa mGC and the mutant protein are immunologically similar; both proteins are linked to the ANF signal in the generation of cyclic GMP synthesis; and in both the ligand binding and catalytic activities are bridged through a defined ATP binding module.

Purification and characterization of the 180-kDa membrane guanylate cyclase containing atrial natriuretic factor receptor from rat adrenal gland and its regulation by protein kinase C

The original concept that cyclic GMP is one of the mediators of the hormone-dependent process of steroidogenesis has been strengthened by the characterization of a 180-kDa protein from rat adrenocortical carcinoma and rat and mouse testes. This protein appears to have an unusual characteristic of containing both the atrial natriuretic factor (ANF)-binding and guanylate cyclase activities, and appears to be intimately involved in the ANF-dependent steroidogenic signal transduction. In rat adrenal glands we now demonstrate: 1) the direct presence of a 180-kDa ANF-binding protein in GTP-affinity purified membrane fraction as evidenced by affinity cross-linking technique and by the Western blot analysis of the partially purified enzyme; 2) that the enzyme is biochemically and immunologically different from the soluble guanylate cyclase as there is no antigenic cross-reactivity of 180-kDa guanylate cyclase antibody with soluble guanylate cyclase; 3) in contrast to the soluble guanylate cyclase, the particulate enzyme is not stimulated by nitrite-generating compounds and hemin; and 4) protein kinase C inhibits both the basal and ANF-dependent guanylate cyclase activity and phosphorylates the 180-kDa guanylate cyclase. These results reveal the presence of a 180-kDa protein in rat adrenal glands and support the contention that: (a) this protein contains both the guanylate cyclase and ANF receptor; (b) the 180-kDa enzyme is coupled with the ANF-dependent cyclic GMP production; (c) the 180-kDa enzyme is biochemically distinct from the nonspecific soluble guanylate cyclase; and (d) there is a protein kinase C-dependent negative regulatory loop for the operation of ANF-dependent cyclic GMP signal pathway which acts via the phosphorylation of 180-kDa guanylate cyclase.

Ubiquitous and bifunctional 180 kDa atrial natriuretic factor-dependent guanylate cyclase

SummaryOriginal studies with rat adrenocortical carcinoma identified a 180 kDa cell-surface protein which contained both guanylate cyclase and atrial natriuretic factor (ANF) receptor, representing a potentially new type of bifunctional receptor protein. It is both a receptor and a guanylate cyclase. This critical conclusion of bifunctionality was based on the observation that the pure 180 kDa protein, whose purity was established by protein staining of the denatured gels, contained both the ligand binding and guanylate cyclase activities. Utilizing the antibody to 180 kDa membrane guanylate cyclase (180 kDa mGC), we now (i) report the complete purification of 180 kDa mGC from rat testes; (ii) demonstrate by affinity cross-linking studies that the homogeneous 180 kDa protein isolated from rat testes and adrenal gland binds ANF and (iii) show that bovine aortic endothelial cell membranes contain the 180 kDa mGC that is ANF-dependent in the production of cyclic GMP. These results validate the conclusion of the bifunctionality, ubiquity, and the general linkage to the ANF-dependent generation of cyclic GMP signal of this protein.

Genetic evidence for ?2-adrenergic receptor subtypes in rat brain, heart and adrenal gland

SummaryA complementary DNA (cDNA) clone - cA2-47 - corresponding to a new α2-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the α2-receptor poly(A+) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5′ half of the coding region of this cDNA at 37°C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet α2-receptor genomic probe failed to hybridize to any rat brain mRNAs.Under lower stringency conditions, hybridization of the full-length cDNA, cA2-47, to selected rat tissue poly(A+) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined. Under the same conditions, a human platelet α2-receptor probe hybridized to similar sized messages of 2.1 and 5.8 kb in rat brain and 2.2 and 6.0 kb in rat heart and adrenal gland. This probe, however, failed to detect the abundant 1.3 kb mRNA common to all tissues or the 3.4 kb message in rat brain. The extent of homology of these messages with cA2-47 is not confined to limited regions of the cDNA since similar hybridization patterns were observed using either 5′-noncoding or 5′-coding regions of the probe.These results provide the first direct evidence of a surprisingly large range of mRNA sizes for members of the α2-receptor family in brain, heart, and adrenal gland. The unique nature of certain members of the family in each of the tissues examined raises the curious possibility that these members might contribute to some of the individualized functions of the brain, cardiovasculature and adrenal gland.

Three immunologically similar atrial natriuretic factor receptors.

One of the atrial natriuretic factor (ANF) receptors is a 180 kDa protein (180 kDa mGC) which possesses the extraordinary characteristic of being bifunctional: it is both a receptor and a guanylate cyclase. In addition to the 180 kDa mGC, there exists another 120–130 kDa protein which is also bifunctional and a 120 kDa disulfide-linked dimeric cell surface protein that is an ANF receptor, but is not a part of guanylate cyclase. A fundamental question that needs to be resolved is: Are these three apparently biochemically distinct ANF receptors structurally similar? With the aid of affinity crosslinking techniques, a highly specific antibody to the 180 kDa mGC, and GTP-affinity techniques, we now demonstrate the presence of three immunologically similar proteins in rat adrenal gland and testes. These proteins migrate as 180 kDa, 130 kDa and 65 kDa under denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis and specifically bind ANF, raising one or more of the following possibilities about their relationships: 1) Degradation of 180 kDa to 130 kDa and 65 kDa occurs during purification; 2) 180 kDa bears a precursor-product relationship with 130 kDa and 65 kDa, suggesting the role of a protease in the processing procedure; 3) these proteins are a result of gene splicing; or 4) they are the products of three separate, but very closely related genes.

Inhibition of α2-adrenergic receptor-mediated cyclic GMP formation by a phorbol ester, a protein kinase C activator

alpha 2-adrenergic receptor-mediated signal transduction in rat adrenocortical carcinoma cells occurs through the opposing regulation of two second messengers, cyclic GMP and cyclic AMP, in which guanylate cyclase is coupled positively and adenylate cyclase negatively to the receptor signal. We now show that in these cells phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, inhibits the alpha 2-agonist (p-aminoclodine)-dependent production of cyclic GMP in a dose-dependent and time-dependent fashion. The half-maximal inhibitory concentration of PMA was 10(-10) M. A protein kinase C inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H-7), caused the release of the PMA-dependent attenuation of p-aminoclodine-stimulated cyclic GMP formation. These results suggest that protein kinase C negatively regulates the alpha 2-receptor coupled cyclic GMP system in these cells, a feature apparently shared with the other cyclic GMP-coupled receptors such as those of muscarine, histamine, and atrial natriuretic factor.