The neurokinin-3 receptor (NK3R) antagonist SB222200 prevents the apomorphine-evoked surface but not nuclear NK3R redistribution in dopaminergic neurons of the rat ventral tegmental area.

Schizophrenia is a severe condition that has been associated with functional abnormalities in dopaminergic (DA) neurons of the ventral tegmental area (VTA). Neurokinin-3 receptors (NK3Rs) of the tachykinin family of neuropeptides modulate the activity of VTA DA neurons and might be involved in DA abnormalities relevant to schizophrenia. Recent work from our lab showed that systemic injection of the dopamine D1/D2 receptor agonist apomorphine in rats, which mimics schizophrenia-like behaviors in humans, also evoked a redistribution of NK3Rs in DA neurons of the rat VTA. In the present study, VTA microinjection of the selective NK3R antagonist SB222200 (1 nmol/0.2 µl) or the nuclear import blocker SN50 (2 µg/0.2 µl) was performed in awake rats 10 min prior to systemic injection of apomorphine. VTA sections were dual immunolabeled for the NK3R (immunogold) and the dopamine synthesizing enzyme tyrosine hydroxylase (TH, immunoperoxidase). Electron microscopic quantifications of somatic and dendritic densities of NK3 immunogold particles were compared in rats receiving central and systemic injections. In DA (TH-labeled) dendrites, VTA microinjection of SB222200 prevented the apomorphine-evoked decrease in surface NK3R density as well as the apomorphine-induced increase in cytoplasmic NK3R density. In contrast, VTA microinjection of SN50, but not SB222200, prevented the apomorphine-induced increase in nuclear NK3R density. VTA microinjection of SB222200 or SN50 without apomorphine had no effect on the NK3R distribution or density in TH and non-TH profiles within the VTA. In non-TH, presumably GABAergic neurons of the VTA, the NK3R densities in somata and dendrites were not significantly changed by apomorphine with or without SB222200. The results suggest that the NK3R antagonist SB222200 is effective against the apomorphine-evoked NK3R internalization in VTA DA dendrites, but does not prevent nuclear NK3R trafficking in VTA DA neurons. These results might have important implications in targeting NK3R antagonists in basic or clinical studies.

Apomorphine-evoked redistribution of neurokinin-3 receptors in dopaminergic dendrites and neuronal nuclei of the rat ventral tegmental area.

Mammalian neurokinin-3 (NK(3)) receptors of the tachykinin family of neuropeptides have been shown to activate dopaminergic neurons of the ventral tegmental area (VTA), a midbrain area displaying dopaminergic dysfunctional activity in schizophrenia. The recent finding of NK(3) receptors in VTA neuronal nucleus highlights a new level of neuromodulation, in addition to the traditional tachykinin-induced NK(3) receptor internalization and activation of second messenger signaling pathways. The function of nuclear NK(3) receptors is still unknown. It is also unclear how dopaminergic activation is affecting the NK(3) receptor distribution in the VTA. In the present study, trafficking of the NK(3) receptor in somatodendritic profiles of dopaminergic and non-dopaminergic neurons of the rat VTA was investigated following acute systemic administration of the dopamine D(1)/D(2) receptor agonist apomorphine. VTA sections were dual immunolabeled for the NK(3) receptor (immunogold) and the dopamine synthesizing enzyme tyrosine hydroxylase (TH, immunoperoxidase). Electron microscopic quantifications of somatic and dendritic densities of NK(3) immunogold particles with or without TH immunolabeling were compared in vehicle-injected or apomorphine-injected rats. In dopaminergic (TH) neurons, apomorphine evoked a significant increase in NK(3) receptor densities in cytoplasmic and nuclear portions of the soma. These changes were accompanied by a respective decrease and increase in plasmalemmal and cytoplamic NK(3) receptor densities in dopaminergic dendrites. In non-TH neurons, presumably GABAergic neurons of the VTA, the NK(3) receptor densities in somata and dendrites were not significantly altered by apomorphine. The results suggest that dopaminergic receptor activation is inducing a rapid mobilization of NK(3) receptors in VTA dopaminergic neurons. The apomorphine-evoked NK(3) receptors plasticity might reflect dendritic internalization and translocation of NK(3) receptors toward the soma and nucleus. This trafficking is not observed in non-dopaminergic neurons of the VTA. The selective apomorphine-evoked redistribution of VTA NK(3) receptors might have important implications in normal or pathological conditions such as schizophrenia.

Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation.

The vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH(2)) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO(2), generating the gamma-carboxylated glutamate product. How the carboxylase effects this conversion is poorly understood because the active site has not been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. (1995) Science 269, 1684-1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH(2)) capable of generating the carbanion. To define the active site and test this model, we identified the amino acids that participate in these reactions. N-ethyl maleimide inhibited epoxidation and carboxylation, and both activities were equally protected by KH(2) preincubation. Amino acid analysis of (14)C- N-ethyl maleimide-modified human carboxylase revealed 1.8-2.3 reactive residues and a specific activity of 7 x 10(8) cpm/hr per mg. Tryptic digestion and liquid chromatography electrospray mass spectrometry identified Cys-99 and Cys-450 as active site residues. Mutation to serine reduced both epoxidation and carboxylation, to 0. 2% (Cys-99) or 1% (Cys-450), and increased the K(m)s for a glutamyl substrate 6- to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine/serine nucleophilicity, a property shared by many active site thiol enzymes. These studies, which represent a breakthrough in defining the carboxylase active site, suggest a revised model in which the glutamyl substrate indirectly coordinates at least one thiol, forming a catalytic complex that ionizes a thiol to initiate KH(2) oxygenation.

Glycosylation sites in the atrial natriuretic peptide receptor: oligosaccharide structures are not required for hormone binding.

Atrial natriuretic peptide (ANP) is a hormone involved in cardiovascular homeostasis through its natriuretic and vasodilator actions. The ANP receptor that mediates these actions is a glycosylated transmembrane protein coupled to guanylate cyclase. The role of glycosylation in receptor signaling remains unresolved. In this study, we determined, by a combination of HPLC/MS and Edman sequencing, the glycosylation sites in the extracellular domain of ANP receptor (NPR-ECD) from rat expressed in COS-1 cells. HPLC/MS analysis of a tryptic digest of NPR-ECD identified five glycosylated peptide fragments, which were then sequenced by Edman degradation to determine the glycosylation sites. The data revealed Asn-linked glycosylation at five of six potential sites. The type of oligosaccharide structure attached at each site was deduced from the observed masses of the glycosylated peptides as follows: Asn13 (high-mannose), Asn180 (complex), Asn306 (complex), Asn347 (complex), and Asn395 (high-mannose and hybrid types). Glycosylation at Asn180 and Asn347 was partial. The role of glycosyl moieties in ANP binding was examined by enzymatic deglycosylation of NPR-ECD followed by binding assay. NPR-ECD deglycosylated with endoglycosidase F2 and endoglycosidase H retained ANP-binding activity and showed an affinity for ANP similar to that of untreated NPR-ECD. Endoglycosidase treatment of the full-length ANP receptor expressed in COS-1 cells also had no detectable effect on ANP binding. These results suggest that, although glycosylation may be required for folding and transport of the newly synthesized ANP receptor to the cell surface, the oligosaccharide moieties themselves are not involved in hormone binding.

Structure of the dimerized hormone-binding domain of a guanylyl-cyclase-coupled receptor.

The atrial natriuretic peptide (ANP) hormone is secreted by the heart in response to an increase in blood pressure. ANP exhibits several potent anti-hypertensive actions in the kidney, adrenal gland and vascular system. These actions are induced by hormone binding extracellularly to the ANP receptor, thereby activating its intracellular guanylyl cyclase domain for the production of cyclic GMP. Here we present the crystal structure of the glycosylated dimerized hormone-binding domain of the ANP receptor at 2.0-A resolution. The monomer comprises two interconnected subdomains, each encompassing a central beta-sheet flanked by alpha-helices, and exhibits the type I periplasmic binding protein fold. Dimerization is mediated by the juxtaposition of four parallel helices, arranged two by two, which brings the two protruding carboxy termini into close relative proximity. From affinity labelling and mutagenesis studies, the ANP-binding site maps to the side of the dimer crevice and extends to near the dimer interface. A conserved chloride-binding site is located in the membrane distal domain, and we found that hormone binding is chloride dependent. These studies suggest mechanisms for hormone activation and the allostery of the ANP receptor.

Atrial natriuretic factor binding to its receptor is dependent on chloride concentration: A possible feedback-control mechanism in renal salt regulation.

Although considerable evidence indicates a role for atrial natriuretic factor (ANF) in renal salt regulation, other studies have found a lack of natriuretic response to high-plasma ANF under certain physiological and pathophysiological conditions. The mechanism for this apparent insensitivity to ANF is unknown. In the present study, it was found that ANF binding to its receptor requires the presence of chloride and occurs in a chloride concentration-dependent manner. ANF binding was measured using the purified recombinant hormone-binding domain of the ANF receptor in the presence of 0.1 mol/L NaCl or other selected salt. High specific binding was detected in the presence of NaCl, KCl, or NH(4)Cl. However, binding was undetectable when the salt was replaced with NaHCO(3), CH(3)COONa, or CH(3)COONH(4), indicating that binding requires the presence of chloride. Chloride dependence was also found with the native receptor in bovine adrenocortical membrane preparations. ANF binding to the recombinant protein was chloride concentration-dependent over a range from 0.05 to 10 mmol/L, and a half-maximum binding was attained at approximately 0.6 mmol/L equivalent chloride concentration. Competitive-binding assays at several fixed concentrations of NaCl showed that lowering chloride concentration caused a decrease in maximum binding but did not alter K(d) values, suggesting that a loss of chloride turns off ANF binding rather than reducing affinity for ANF. Saturation-binding studies showed that excess ANF cannot overcome loss of binding caused by low chloride. Chloride-dependent ANF-receptor binding may function as a feedback-control mechanism regulating the ANF-receptor action and, hence, renal sodium excretion.

Disulfide bond structure of the atrial natriuretic peptide receptor extracellular domain: conserved disulfide bonds among guanylate cyclase-coupled receptors.

The disulfide bond structure of the extracellular domain of rat atrial natriuretic peptide (ANP) receptor (NPR-ECD) has been determined by mass spectrometry (MS) and Edman sequencing. Recombinant NPR-ECD expressed in COS-1 cells and purified from the culture medium binds ANP with as high affinity as the natural ANP receptor. Reaction with iodoacetic acid yielded no S-carboxymethylcysteine, indicating that all six Cys residues in NPR-ECD are involved in disulfide bonds. Electrospray ionization MS of NPR-ECD deglycosylated by peptide-N-glycosidase F gave a molecular mass of 48377.5+/-1.6 Da, which was consistent with the presence of three disulfide bonds. Liquid chromatography MS analysis of a lysylendopeptidase digest yielded three cystine-containing fragments with disulfide bonds Cys(60)-Cys(86), Cys(164)-Cys(213) and Cys(423)-Cys(432) based on their observed masses. These bonds were confirmed by Edman sequencing of each of the three fragments. No evidence for an inter-molecular disulfide bond was found. The six Cys residues in NPR-ECD, forming a 1-2, 3-4, 5-6 disulfide pairing pattern, are strictly conserved among A-type natriuretic peptide receptors and are similar in B-type receptors. We found that in other families of guanylate cyclase-coupled receptors, the Cys residues involved in 1-2 and 5-6 disulfide pairs are conserved in nearly all, suggesting an important contribution of these disulfide bonds to the receptor's structure and function.

A study of the ability of tissue plasminogen activator to diffuse into the subretinal space after intravitreal injection in rabbits.

PURPOSE: Intravitreal injections of tissue plasminogen activator have been used to lyse fibrin from blood in the subretinal space, despite the lack of proof that tissue plasminogen activator can diffuse across the retina. We tested whether tissue plasminogen activator injected into the vitreous could penetrate the neural retina and enter the subretinal space. METHODS: We injected a mixture of 50 microg of tissue plasminogen activator (70 kD) labeled with fluorescein isothiocyanate and rhodamine B isothiocyanate-labeled dextran, which has a lower molecular weight (20 kD), into the midvitreous cavity of one eye in each of 18 rabbits. The eyes were enucleated after 3, 6, and 24 hours, and cryosections were examined with epifluorescent microscopy to determine the distribution of the labeled molecules. We also evaluated tissue plasminogen activator pharmacokinetics in one eye each of 18 rabbits in which a subretinal clot was induced by injecting autologous blood (50 microL) into the subretinal space through the sclera. Fluorescein isothiocyanate-labeled tissue plasminogen activator was injected into the vitreous 2 days after induction of the subretinal clot. RESULTS: Fluorescein isothiocyanate-labeled tissue plasminogen activator was present at the vitreal surface of the retina in a linear array in all 36 eyes studied, whereas the rhodamine B isothiocyanate-labeled dextran had diffused throughout the neural retina in the same sections. No fluorescein isothiocyanate signal was observed in the neural retina or in the subretinal clot. Vitreous hemorrhage caused by retinal perforation was observed in all eyes with intraretinal hemorrhage in which fluorescein isothiocyanate fluorescence was seen in the neural retina and inside the clot. CONCLUSION: Intravitreal tissue plasminogen activator did not diffuse through the intact neural retina to reach a subretinal clot. This study demonstrates no scientific rationale for the intravitreal tissue plasminogen activator treatment of submacular hemorrhage without vitreous hemorrhage presumably caused by an overlying retinal break.

Ligand binding-dependent limited proteolysis of the atrial natriuretic peptide receptor: juxtamembrane hinge structure essential for transmembrane signal transduction.

The atrial natriuretic peptide (ANP) receptor is a 130-kDa transmembrane protein containing an extracellular ANP-binding domain, a single transmembrane sequence, an intracellular kinase-homologous domain, and a guanylate cyclase (GCase) domain. We observed that the receptor, when bound with ANP, was rapidly cleaved by endogenous or exogenously added protease to yield a 65-kDa ANP-binding fragment. No cleavage occurred without bound ANP. This ligand-induced cleavage abolished GCase activation by ANP. Cleavage occurred in an extracellular, juxtamembrane region containing six closely spaced Pro residues and a disulfide bond. Such structural features are shared among the A-type and B-type ANP receptors but not by ANP clearance receptors. The potential role of the hinge structure was examined by mutagenesis experiments. Mutation of Pro(417), but not other Pro residues, to Ala abolished GCase activation by ANP. Elimination of the disulfide bond by Cys to Ser mutations yielded a constitutively active receptor. Pro(417), and Cys(423) and Cys(432) forming the disulfide bond are strictly conserved among GCase-coupled receptors, while other residues are largely variable. The conserved Pro(417) and the disulfide bond may represent a consensus signaling motif in the juxtamembrane hinge structure that undergoes a marked conformational change upon ligand binding and apparently mediates transmembrane signal transduction.

Expression and purification of the extracellular ligand-binding domain of the atrial natriuretic peptide (ANP) receptor: monovalent binding with ANP induces 2:2 complexes.

The receptor for atrial natriuretic peptide (ANP) is a type-I transmembrane protein containing an extracellular ligand-binding domain, a single transmembrane sequence, an intracellular kinase-homologous domain, and a guanylate cyclase (GCase) domain. Binding of ANP to the extracellular domain causes activation of the GCase domain by an as yet unknown mechanism. To facilitate studies of the receptor structure and signaling mechanism, we have expressed the extracellular ANP-binding domain of rat ANP receptor (NPR-ECD) in a water-soluble form. NPR-ECD was purified to homogeneity by ANP-affinity chromatography. SDS-PAGE gave a single 61-kDa band, which coincided with a radioactive band obtained by photoaffinity-labeling with N4alpha-azidobenzoyl-125I-ANP(4-28). Edman degradation gave a single amino-terminal sequence expected for the mature protein. Both trifluoromethanesulfonic acid and peptide-N-glycosidase F treatments yielded a 50-kDa band, indicating N-glycosylation. The molecular mass of 57 725 Da determined by mass spectrometry indicates the carbohydrate content at 16%. NPR-ECD bound ANP with an affinity comparable to that of the full-length receptor. The ligand selectivity of NPR-ECD (in the order ANP > brain natriuretic peptide >> C-type natriuretic peptide) was also similar to that of the full-length receptor. HPLC gel filtration of NPR-ECD gave a peak with an apparent mass of 74 kDa. Preincubation with ANP generated a new 150-kDa peak with a concomitant decrease of the 74-kDa peak. This shift in peak positions was ANP concentration-dependent and was complete at the NPR-ECD-to-ANP molar ratio of 1:1, indicating equimolar binding. The change in the apparent native molecular weight from 74 to 150 kDa suggests that binding causes dimerization of the NPR-ECD:ANP complex to yield an [NPR-ECD:ANP]2 complex.

SPACR, a novel interphotoreceptor matrix glycoprotein in human retina that interacts with hyaluronan.

SPACR (sialoprotein associated with cones and rods), is the major 147-150-kDa glycoprotein present in the insoluble interphotoreceptor matrix of the human retina. Immunocytochemistry localizes SPACR to the matrix surrounding rods and cones (Acharya, S., Rayborn, M. E., and Hollyfield, J. G. (1998) Glycobiology 8, 997-1006). From affinity-purified SPACR, we obtained seven peptide sequences showing 100% identity to the deduced sequence of IMPG1, a purported chondroitin 6-sulfate proteoglycan core protein, which binds peanut agglutinin and is localized to the interphotoreceptor matrix. We show here that SPACR is the most prominent 147-150-kDa band present in the interphotoreceptor matrix and is the gene product of IMPG1. SPACR is not a chondroitin sulfate proteoglycan, since it is not a product of chondroitinase ABC digestion and does not react to a specific antibody for chondroitin 6-sulfate proteoglycan. Moreover, the deduced amino acid sequence reveals no established glycosaminoglycan attachment site. One hyaluronan binding motif is present in the predicted sequence of SPACR. We present evidence that SPACR has a functional hyaluronan binding domain, suggesting that interactions between SPACR and hyaluronan may serve to form the basic macromolecular scaffold, which comprises the insoluble interphotoreceptor matrix.

The human urinary epidermal growth factor (EGF) precursor. Isolation of a biologically active 160-kilodalton heparin-binding pro-EGF with a truncated carboxyl terminus.

In this report, we describe the isolation from human urine of a predominant 160-kDa epidermal growth factor (EGF)-immunoreactive glycoprotein that exhibits affinity for heparin. The purification procedure involved concentration and dialysis of 20-30-liter batches of fresh urine on a high capacity ultrafiltration apparatus followed by chromatography on DEAE-Sephacel, heparin-agarose, and Sephacryl S-300. A nearly homogeneous preparation of 160-kDa protein was obtained with a yield of approximately 1 mg of 160-kDa protein from 25 liters of urine. The amino-terminal sequence of the purified 160-kDa protein, H2N-SAPQHXSXPEGTXA-, matched residues 21-34 of the predicted sequence of human prepro-EGF and established that the 160 kDa protein (pro-EGF) is a product of the prepro-EGF gene. Characterization of the carboxyl terminus of the purified protein by digestion with carboxypeptidase B and by immunoblotting with antisera against synthetic carboxyl-terminal and juxtatransmembrane peptides of prepro-EGF indicated that the carboxyl terminus has been truncated at an arginine residue that corresponds, most likely, to the carboxyl-terminal arginine of the EGF moiety. The intact 160-kDa pro-EGF is biologically active as evidenced by its specific binding to the EGF receptor and activation of the EGF receptor tyrosine kinase in A-431 cell membranes. Purified pro-EGF competitively inhibited the binding of 125I-EGF to human fibroblasts, and it stimulated the proliferation of these cells in culture. When immobilized onto culture dishes, the heparin-binding pro-EGF appeared to function both as an adhesion molecule and as a growth factor for serum-free mouse embryo cells.

High-yield affinity alkylation of the atrial natriuretic factor receptor binding site.

To facilitate characterization of the atrial natriuretic factor (ANF) receptor, we have developed an affinity labeling procedure, stepwise affinity labeling, which allows specific labeling of ANF binding sites in adrenal plasma membranes at high yields. An iodoacetyl (IAc-), bromoacetyl (BrAc-), or maleimidobenzoyl group was attached to the amino-terminal alpha-amino group of the ANF(4-28) peptide, and the peptide derivatives were radioiodinated at Tyr-28 to obtain affinity reagents, N4alpha-IAc-[125I]ANF(4-28), N4alpha-BrAc-[125I]ANF(4-28), and N4alpha-(maleimidobenzoyl)-[125I]ANF(4-28). Receptor labeling was carried out in a stepwise fashion as follows: (1) Membranes were treated with p-chloromercuriobenzenesulfonic acid (PCMBS) or N-ethylmaleimide to block sulfhydryl groups; (2) the affinity reagent was allowed to bind to the receptor at 0 degrees C for 1 h; and (3) the membranes were washed to remove unbound reagent and were incubated at room temperature to effect alkylation reaction. Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography revealed specific labeling of a 130-kDa ANF receptor. On the basis of 125I-radioactivity incorporated, the labeling yields were estimated to be 70%, 52%, and 21% for the reactions with IAc-[125I]ANF(4-28), BrAc-[125I]ANF(4-28), and (maleimidobenzoyl)-[125I]ANF(4-28), respectively. The efficiency of receptor labeling by the stepwise procedure using IAc-[125I]ANF(4-28) was 27-fold greater than that obtained by photoaffinity labeling using N3Bz-[125I]ANF(4-28) and 63-fold greater than that by direct cross-linking using disuccinimidylsuberate and [125I]ANF(4-28) under comparable conditions. Digestion of the membrane protein labeled with IAc-[125I]ANF(4-28) by BrCN, endoproteinase Glu-C, and endoproteinase Lys-C gave single radiolabeled bands with apparent masses of 40, 18, and 29 kDa, respectively. Reversed-phase HPLC separation of the digests also gave single major peaks. The confinement of the affinity label to one major fragment in each digest suggests that the cross-linking occurred at a single or a limited number of sites. The stepwise affinity labeling with the high cross-linking yield and specificity may be useful for analyzing the ANF receptor binding site structure.

Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function.

The alpha 1-adrenergic receptors activate a phospholipase C enzyme by coupling to members of the large molecular size (approximately 74 to 80 kilodaltons) G alpha h family of guanosine triphosphate (GTP)-binding proteins. Rat liver G alpha h is now shown to be a tissue transglutaminase type II (TGase II). The transglutaminase activity of rat liver TGase II expressed in COS-1 cells was inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) or by alpha 1-adrenergic receptor activation. Rat liver TGase II also mediated alpha 1-adrenergic receptor stimulation of phospholipase C activity. Thus, G alpha h represents a new class of GTP-binding proteins that participate in receptor signaling and may be a component of a complex regulatory network in which receptor-stimulated GTP binding switches the function of G alpha h from transglutamination to receptor signaling.

Secretion of thermostable DNA polymerase using a novel baculovirus vector.

Baculovirus-mediated expression has become a powerful tool for the high yield production of functionally active recombinant proteins. In order to further enhance the utility of this expression system, we constructed versatile transfer vectors that facilitate the secretion of recombinant proteins from host insect cells by inserting functional secretory leader sequences down-stream of the polyhedrin promoter. In-frame insertion of cDNA sequences results in the synthesis of fusion proteins containing a heterologous signal sequence which directs the recombinant protein to the secretory pathway. Human and insect leader sequences were successfully tested with a number of proteins including the thermostable Pyrococcus furiousus (Pfu) DNA polymerase in an effort to maximize secretion of heterologous proteins from insect cells. The human placental alkaline phosphatase signal sequence (MLGPCMLLLLLLLGLRLQLSLG) proved to be optimal for the secretion of not only this thermostable bacterial enzyme but also for the secretion of other biologically active polypeptides.

Regulated cleavage-secretion of the membrane-bound angiotensin-converting enzyme.

Angiotensin-converting enzyme (ACE) is an ectoprotein anchored in the plasma membrane through a hydrophobic domain near its carboxyl-terminal region. Mouse epithelial cells transfected with rabbit testicular ACE cDNA, synthesize, glycosylate, and secrete ACE by cleavage processing of its membrane-anchoring carboxyl-terminal region. Because the cleavage-secretion process is slow, the enzyme accumulates on the cell surface. We show that this process can be enhanced by treatment of cells with tumor-promoting phorbol esters leading to depletion of the cell surface enzyme. The cleavage processing occurs only after the protein has reached the cell surface and is not affected by disruption of the Golgi apparatus or the lysosomal compartments. The exact peptide bond cleaved has been identified by sequencing the amino-terminal residues of the purified COOH-terminal tail left in the cells after ACE is secreted and the carboxyl-terminal residues of secreted ACE. The cleavage occurs at a monobasic site between Arg-663 and Ser-664 generating the soluble enzyme and leaving a cell-bound protein of 74 residues. These results demonstrate the existence of cellular mechanisms that regulate the conversion of cell-bound ACE to a soluble enzyme.

Aortic smooth muscle contains guanylate-cyclase-coupled 130-kDa atrial natriuretic factor receptor as predominant receptor form. Spontaneous switching to 60-kDa C-receptor upon cell culturing.

Photoaffinity labeling of atrial natriuretic factor (ANF) receptor in the plasma membranes from bovine aortic smooth muscle tissue using N alpha 5-(4-azidobenzoyl)-ANF-(5-28)- peptide labeled with 125I yielded a 130-kDa band. However, when smooth muscle cells from the same bovine aorta were placed in culture, the 130-kDa receptor quickly disappeared and a 60-kDa band began to appear at high density. After three passages, essentially no 130-kDa band was found and only the 60-kDa band was strongly labeled. The primary structures of the two receptor forms were compared by radiochemical peptide mapping after endoproteinase Glu-C digestion of photoaffinity-labeled and detergent-solubilized 130-kDa receptor from the aorta or the 60-kDa receptor from the cultured cells. The peptide mapping showed courses of digestion that were significantly different from each other, suggesting difference in their primary structures. The basal guanylate cyclase activity in the aortic membranes was 1.0 pmol cGMP produced.min-1.mg protein-1 at 37 degrees C using Mn(2+)-GTP as substrate. The corresponding activity in the membranes from the cultured cells was 20 fmol cGMP.min-1.mg protein-1. Binding studies gave a density of binding sites (Bmax) of 82 fmol/mg protein for the aortic membranes and 850 fmol/mg protein for the cultured cell membranes. These data suggest that the major form of ANF receptor in the cultured cells, namely the 60-kDa receptor, lacked guanylate cyclase activity. Northern blot analysis of poly(A)-RNA extracted form bovine thoracic aorta or adrenal cortex gave a single 3.6-kb band when 32P-labeled human A-type ANF receptor cDNA was used as a hybridization probe. However, no band was detected when C-receptor cDNA was used as a probe. In addition to the major 130-kDa band, extended SDS/PAGE revealed two additional faint bands with estimated molecular masses of 126 kDa and 135 kDa. Treatment with endoglycosidase H resulted in disappearance of the 126-kDa band and appearance of a 100-kDa band. The 130-kDa and 135-kDa bands were unchanged. Treatment by endoglycosidase F or glycopeptidase F reduced all three bands to a single 100-kDa band. These results suggest that the slight difference in mobility is due to different states of glycosylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Myotrophin in human cardiomyopathic heart.

Earlier, myotrophin, a factor, has been isolated, purified, and partially sequenced from spontaneously hypertensive rat hearts that stimulated myocyte growth. To evaluate the role of myotrophin in the initiation of the human dilated cardiomyopathic heart, we have isolated and purified myotrophin to homogeneity (approximately 50,000-fold) as defined by reverse-phase high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). During purification, we used a bioassay system in which adult myocardial cells maintained in culture were used to evaluate protein synthesis by the incorporation of [3H]leucine into myocyte protein. Myotrophin purified from human dilated cardiomyopathic hearts is composed of a single polypeptide chain having an apparent molecular mass of 12 kD, determined by SDS-PAGE. The partial internal amino acid sequence of human myotrophin is very similar to that of rat myotrophin peptide T9. Using a rat myotrophin peptide (T26) antibody, we identified human myotrophin on an immunoblot. These results showed that human myotrophin possesses the T9 and T26 regions of rat myotrophin. Human myotrophin stimulated myocardial protein synthesis and cell growth, similar to the way in which rat myotrophin stimulated these factors. Western blot analysis showed the presence of myotrophin in both dilated cardiomyopathic and normal human hearts. In addition, we observed significantly elevated levels of myotrophin in dilated cardiomyopathic human hearts when compared with age- and sex-matched normal control hearts. From these observations, we conclude that myotrophin is present in normal human hearts, is found at higher levels in dilated cardiomyopathic human hearts, and may play a role in the initiation of cardiac hypertrophy as well as in normal growth of cardiac myocytes in humans.

Proteolytic cleavage of atrial natriuretic factor receptor in bovine adrenal membranes by endogenous metalloendopeptidase. Effects on guanylate cyclase activity and ligand-binding specificity.

Atrial natriuretic factor (ANF) is a peptide hormone from the heart atrium with potent natriuretic and vasorelaxant activities. The natriuretic activity of ANF is, in part, mediated through the adrenal gland, where binding of ANF to the 130-kDa ANF receptor causes suppression of aldosterone secretion. Incubation of bovine adrenal membranes at pH < 5.6 caused a rapid and spontaneous cleavage of the 130-kDa ANF receptor, yielding a 65-kDa polypeptide that could be detected by photoaffinity labeling by 125I-labeled N alpha 4-azidobenzoyl-ANF(4-28) followed by SDS/PAGE under reducing conditions. Within 20 min of incubation at pH 4.0, essentially all the 130-kDa receptor was converted to a 65-kDa ANF binding protein. This cleavage reaction was completely inhibited by inclusion of 5 mM EDTA. When SDS/PAGE was carried out under non-reducing conditions, the apparent size of the ANF receptor remained unchanged at 130 kDa, indicating that the 65-kDa ANF-binding fragment was still linked to the remaining part(s) of the receptor polypeptide through a disulfide bond(s). The disappearance of the 130-kDa receptor was accompanied by a parallel decrease in guanylate cyclase activity in the membranes. Inclusion of EDTA in the incubation not only prevented cleavage of the 130-kDa receptor, but also protected guanylate cyclase activity, indicating that proteolysis, but not the physical effects of the acidic pH, causes inactivation of guanylate cyclase. The 130-kDa ANF receptor in adrenal membranes was competitively protected from photoaffinity labeling by ANF(1-28) or ANF(4-28), but not by atriopeptin I [ANF(5-25)] or C-ANF [des-(18-22)-ANF(4-23)-NH2]. On the contrary, the 65-kDa ANF-binding fragment generated after incubation at pH 4.0 was protected from labeling by any of the above peptides, indicating broader binding specificity. After incubation in the presence of EDTA, the 130-kDa ANF receptor, which was protected from proteolysis, retained binding specificity identical to that of the 130-kDa receptor in untreated membranes. The results indicate that the broadening of selectivity is caused by cleavage, but not by the physical effect of acidic pH. Spontaneous proteolysis of ANF receptor by an endogenous metalloendopeptidase, occurring with concomitant inactivation of guanylate cyclase activity and broadening of ligand-binding selectivity, may be responsible for the generation of low-molecular-mass receptors found in the adrenal gland and other target organs of ANF. The proteolytic process may play a role in desensitization or down-regulation of the ANF receptor.

Cloning of the gene and cDNA for human heart chymase.

We have recently identified and characterized a chymotrypsin-like serine proteinase in human heart (human heart chymase) that is the most catalytically efficient enzyme described, thus far, for the cleavage of angiotensin I to yield angiotensin II and the dipeptide His-Leu. Compared to other chymases, this enzyme also has an unusually high degree of specificity for the substrate angiotensin I. We report here the molecular cloning and nucleotide sequence of the gene and cDNA encoding human heart chymase, and determination of its entire deduced amino acid sequence. These data indicate that human heart chymase is highly homologous to other members of the chymase subfamily of chymotrypsin-like proteinases and, most likely, all evolved from a common ancestral gene. Potential regulatory elements found in the 5'-untranslated region of other chymases are also found in the human heart chymase gene. However, this gene lacks mast cell-specific sequences found in the 5'- and 3'-untranslated regions of the rat chymase II gene. In addition, human heart chymase contains clusters of unique amino acid sequences located at key positions likely involved in substrate binding, which may contribute to its high substrate specificity. These contrasting features of the human heart chymase gene and cDNA, and the potential determinants of its primary structure that underlie its unique functional characteristics are considered.