[Interaction of the beta1-subunit of G-proteins with the actin cytoskeleton]. / Vzaimodeistvie beta1-sub''edinits G-belkov s aktinovym tsitoskeletom.

Using immunoblotting with specific antibodies, we have identified beta 1- and beta 2-subunits of Gi-proteins in membrane and cytosolic fractions of pig lung. It has been shown that beta 1-subunit is present both in membrane and cytosolic fractions, whereas beta 2-subunit is associated only with membranes. Activation of membrane-bound G proteins with non-hydrolysable GTP analogues have led to partial release from membrane of beta 1-, but not beta 2-subunits. When depolymerisation of F-actin during fractionation was prevented, both beta 1- and beta 2-subunits were found in fraction containing total membranes and F-actin. Dialysis of this fraction into low ionic strength buffer caused depolymerization of the bulk of actin and release of about 1/4 of beta 1-subunits into solution. The data presented here suggest that distribution of beta 1-subunits between membrane and cytosol could depend on the state of actin cytoskeleton.

Atrial natriuretic peptide modulates alveolar type 2 cell adenylyl and guanylyl cyclases and inhibits surfactant secretion.

Alveolar epithelial type 2 (T2) cells isolated from the lungs of adult rats responded to exogenous atrial natriuretic peptide (ANP) by two signalling mechanisms. First, ANP induced a dose-dependent reduction of ligand-stimulated adenylyl cyclase activity and cAMP accumulation. This effect was inhibited by the addition of GDPbetaS or by pretreatment with pertussis toxin (PT), consistent with mediation by a Gi protein(s). PT-catalyzed [32P]ADP-ribosylation, immunoblots with specific antisera, and Northern blot analysis demonstrated that T2 cells contain the G-proteins Gi2 and Gi3 which could transduce this signal. ANP also promoted PT-insensitive, dose-dependent accumulation of cGMP, consistent with activation of a receptor guanylyl cyclase. Isoproterenol-stimulated phosphatidylcholine secretion was markedly attenuated by ANP, and this effect was inhibited by PT pretreatment, consistent with mediation by a Gi protein(s). These data indicate that in addition to the lung being a major clearance organ for circulating ANP, lung parenchymal cells are targets of ANP action.

[Signal-conducting and low molecular weight GTP-binding proteins from the lung and endothelium: localization in membranes and cytosol, interaction with F-actin]. / Signal-provodiashchie i nizkomolekuliarnye GTP-sviazyvaiushchie belki legkikh i éndoteliia: lokalizatsiia v membranakh i tsitozole, vzaimodeistvie s F-aktinom.

The following proteins have been identified in mammalian lung and endothelium, using [32P]ADP-ribosylation by bacterial ADP-ribosyltransferase, immuno- and [alpha-32P]GTP-blottings: 41 kDa Gi1 alpha, 40 kDa Gi2 alpha, 41 kDa Gi3 alpha, 40 kDa and 45 kDa subunits of GS alpha, 36 kDa beta 1 and 35 kDa beta 2 subunits of signal-transmitting GTP-binding proteins (G-proteins), the 19-26 kDa low molecular weight GTP-binding proteins (SMG-proteins) ras, rho, rac, G25K (Gp), as well as ARF and SMG proteins binding with a high affinity to [alpha-32P]GTP. These G- and SMG-proteins are contained in various proportions in membrane and cytosol fractions of lung and endothelium cells. Subunits Gi2 alpha and GS alpha (but not beta 1 or SMG-proteins) my partially (approximately 1%) dissociate from the membrane by the action of the GTP analogs GTP[S] or Gpp(NH)p in the presence of magnesium ions. Extraction with low ionic strength buffer solutions in the presence of EDTA is accompanied by the release of G-actin sensitive to whooping cough toxin Gi2 alpha and beta i subunits. The functionally coupled into a alpha beta gamma heterodimer Gi-protein subunits (predominantly Gi2 alpha and beta i) present in the cytosol fraction as well as the SMG-proteins revealed by [alpha-32P]GTP-blotting (but not the SMG-proteins sensitive to the botulinic C3 exoenzyme, rho/rac, or ARF, may interact with F-actin. Approximately 20% of these proteins are associated with the Triton X-100 insoluble (cytoskeletal) fraction of the endothelium. A conclusion is drawn that interactions of G- and SMG-proteins with actin filaments may be the reason for the formation of "multidisperse" structure in a cell.

Apparent activation of rabbit lung membrane adenylate cyclase by cytosolic proteins possessing adenylate kinase activity.

Lung cytosolic fraction (23500 x g supernatant) activates cAMP synthesis by lung membrane adenylate cyclase (AC). 23 kDa and 29 kDa proteins were isolated from rabbit lung cytosolic fraction in a homogeneous state, as 'activators' of lung membrane AC. Both of these proteins possess high adenylate kinase (AK) activity and are able to mimic the 'activating' effect of lung cytosol on the lung membrane AC in the standard incubation mixture devoid of adenylate kinase. The activating effect is abolished in the presence of adenylate kinase inhibitor DAPP and after heat- or trypsin-treatment of the cytosolic fraction. Commercial adenylate kinase or nonionic detergent Lubrol PX activate cAMP synthesis by lung membrane AC in a similar manner to that of cytosolic fraction. In the presence of commercial adenylate kinase or Lubrol PX no activating effect of the cytosolic fraction on lung membrane AC is revealed. The ability of cytosolic fraction, commercial adenylate kinase, Lubrol PX or purified 23 kDa and 29 kDa proteins to activate cAMP synthesis by lung membrane AC correlates with their ability to support the constant ATP (AC substrate) concentration in the AC assay mixture. Our data indicate that 'activation' of lung membrane AC in the presence of cytosolic fraction may be produced by cytosolic adenylate kinase activity which regenerates ATP from AMP in the presence of creatine kinase and creatine phosphate providing the substrate for cAMP synthesis by AC.

Signal-transducing GTP-binding proteins of mammalian heart and lungs.

Signal-transducing GTP-binding Proteins of Mammalian Heart and Lungs. Journal of Molecular and Cellular Cardiology (1989) 21 (Suppl I) 91-95. Three distinct G-proteins have been found in mammalian heart sarcolemma: Gi (alpha i = 40 kDa, beta = 36 kDa, and lambda less than 14 kDa), Gp (alpha p = 23 kDa, beta = 36 kDa, and lambda less than 14 kDa), and Gs (alpha s = 42 kDa). ADP-ribosylation of sarcolemmal alpha i by pertussis toxin (PT) or preincubation of sarcolemma with protein kinase C and PMA resulted in increased adenylate cyclase activity and blockade of GTP-dependent inhibition by carbachol whereas the GTP-dependent activating effect of isoproterenol on the adenylate cyclase was preserved. ADP-ribosylation of alpha i in sarcolemma by endogenous NADP-sensitive ADP-ribosyltransferase abolished the PT-induced ADP-ribosylation of alpha i. Gpp (NH)p attenuated the PT-induced ADP-ribosylation of alpha i and promoted the cholera toxin (CT)-induced ADP-ribosylation of alpha s. The CT-induced alpha s ADP-ribosylation was enhanced in the presence of heart cytosol. Soluble Gi- and Gs-proteins were identified in lung cytosol. The 40 kDa alpha i in membrane and soluble fractions was ADP-ribosylated by PT, while the soluble 42 kDa alpha s was ADP-ribosylated by CT in lung tissue. The ADP-ribosylation of soluble alpha i by PT-suppressed guanyl nucleotide binding to Gi. The apparent molecular mass of partially purified soluble Gi was 75 kDa.