The effect of detergents on trimeric G-protein activity in isolated plasma membranes from rat brain cortex: correlation with studies of DPH and Laurdan fluorescence.

The effect of non-ionic detergents on baclofen (GABAB-R agonist)-stimulated G-protein activity was measured as a [(35)S]GTPgammaS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic--a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABAB-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58>Triton X-100>Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (rDPH) incorporated into the hydrophobic PM interior. Decrease of rDPH, in the order of Brij58>Triton X-100>Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time phi paralleled by an increase of diffusion wobbling constant Dw (analysis by time-resolved fluorescence according to "wobble-in-cone" model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58>Triton X-100>Digitonin.

Subcellular redistribution of trimeric G-proteins--potential mechanism of desensitization of hormone response: internalization, solubilization, down-regulation.

Agonist-induced subcellular redistribution of G-protein coupled receptors (GPCR) and of trimeric guanine-nucleotide binding regulatory proteins (G-proteins) represent mechanisms of desensitization of hormone response, which have been studied in our laboratory since 1989. This review brings a short summary of these results and also presents information about related literature data covering at least small part of research carried out in this area. We have also mentioned sodium plus potassium dependent adenosine triphosphatase (Na, K-ATPase) and 3H-ouabain binding as useful reference standard of plasma membrane purity in the brain.

Biochemistry of transmembrane signaling mediated by trimeric G proteins.

Many extracellular signals are at the cell surface received by specific receptors, which upon activation transduce information to the appropriate cellular effector molecules via trimeric G proteins. The G protein-mediated cascades ultimately lead to the highly refined regulation of systems such as sensory perception, cell growth, and hormonal regulation. Transmembrane signaling may be seriously deranged in various pathophysiological conditions. Over the last two decades the major experimental effort of our group has been devoted to better understanding the molecular mechanisms underlying transmembrane signaling regulated by G proteins and to the closely related process of desensitization of hormone response. This review provides general information about the basic principles of G protein-regulated transmembrane signaling as well as about our contribution to the current progress in the field.

Membrane-Bound and cytosolic forms of heterotrimeric G proteins in young and adult rat myocardium: influence of neonatal hypo- and hyperthyroidism.

Membrane and cytosolic fractions prepared from ventricular myocardium of young (21-day-old) hypo- or hyperthyroid rats and adult (84-day-old) previously hypo- or hyperthyroid rats were analyzed by immunoblotting with specific anti-G-protein antibodies for the relative content of Gs alpha, Gi alpha/Go alpha, Gq alpha/G11 alpha, and G beta. All tested G protein subunits were present not only in myocardial membranes but were at least partially distributed in the cytosol, except for Go alpha2, and G11 alpha. Cytosolic forms of the individual G proteins represented about 5-60% of total cellular amounts of these proteins. The long (Gs alpha-L) isoform of Gs alpha prevailed over the short (Gs alpha-S) isoform in both crude myocardial membranes and cytosol. The Gs alpha-L/Gs alpha-S ratio in membranes as well as in cytosol increased during maturation due to a substantial increase in Gs alpha-L. Interestingly, whereas the amount of membrane-bound Gi alpha/Go alpha and Gq alpha/G11 alpha proteins tend to lower during postnatal development, cytosolic forms of these G proteins mostly rise. Neonatal hypothyroidism reduced the amount of myocardial Gs alpha and increased that of Gi alpha/Go alpha proteins. By contrast, neonatal hyperthyroidism increased expression of Gs alpha and decreased that of Gi alpha and G11 alpha in young myocardium. Changes in G protein content induced by neonatal hypo- and hyperthyroidism in young rat myocardium were restored in adulthood. Alterations in the membrane-cytosol balance of G protein subunits associated with maturation or induced by altered thyroid status indicate physiological importance of cytosolic forms of these proteins in the rat myocardium.

Differentiation of cultured brown adipocytes is associated with a selective increase in the short variant of g(s)alpha protein. Evidence for higher functional activity of g(s)alphaS.

In order to examine whether the differentiation process in brown adipocytes cultivated in primary culture is associated with substantial alterations in the complement of G proteins, the levels of these proteins were investigated with immuno-electrophoretic techniques in membrane preparations from proliferating and differentiated cultured mouse brown adipocytes. We observed that differentiation was associated with a dramatic (more than threefold) increase in the short variant of G(s)alpha protein (G(s)alphaS). The long variant of G(s)alpha (G(s)alphaL), as well as G(i)1alpha, G(i)2alpha, G(q)alpha, G(11)alpha and Gbeta subunit proteins remained unchanged whereas G(i)3alpha protein was decreased. These changes were accompanied by marked increase in isoprenaline-, forskolin- as well as manganese-stimulated adenylyl cyclase. Thus, the marked increase in beta-adrenergic responsiveness of fully differentiated confluent brown adipocytes (day 8-9), as compared with that of proliferating undifferentiated cells of 'fibroblast phenotype' (day 3-4), is associated with a significant increase in the relative proportion between the short and long variants of G(s)alpha (the G(s)alphaS/G(s)alphaL ratio) along with a decrease in G(i)3alpha protein. These data also suggest that the short variant of G(s)alpha exhibits higher functional activity than the long variant of this G protein.

Resolution and identification of Gq/G11alpha and Gialpha/Goalpha proteins in brown adipose tissue: effect of cold acclimation.

Levels of guanine nucleotide-binding proteins G(q)alpha and G(11)alpha, which produce receptor regulation of phospholipase C, were measured immunologically in purified plasma membrane fractions of hamster brown adipose tissue (BAT). This was achieved by immunoblotting with antisera (CQ series) that identify these two polypeptides equally, following separation of the plasma membranes using SDS-PAGE in the presence of 6 M urea, i.e. conditions that can resolve G(q)alpha and G(11)alpha. The ratio of levels of G(q)alpha to G(11)alpha was 1:1. A similar approach was used for resolution and identification of G(o)1alpha and G(o)2alpha, the latter representing the prevailing form of G(o)alpha proteins in this tissue. Although clearly recognized in brain microsomes, which were used as positive controls, no detected levels of G(o)*alpha protein were noted. Using specific anti-peptide antibodies directed against the carboxy-terminal decapeptide of G(i)3alpha, this G protein was also found to be expressed in BAT tissue. Cold acclimation resulted in reduction of the plasma membrane levels of all these Galpha proteins.

G proteins, beta-adrenoreceptors and beta-adrenergic responsiveness in immature and adult rat ventricular myocardium: influence of neonatal hypo- and hyperthyroidism.

Thyroid hormones influence a wide range of physiological responses and the heart is considered a major target organ for triiodothyronine action. In the present study we examined closely the presumed relationship between altered thyroid status in the newborn rat and maturation of the regulatory components of the myocardial hormone-sensitive adenylyl cyclase signaling system. Beta -adrenoceptors and the alpha subunits of the stimulatory (Gs) as well as inhibitory (Gi) G proteins were determined in ventricular myocardium of immature (21-day-old) hypo- or hyperthyroid rats and in adult (84-day-old) previously hypo- or hyperthyroid rats. The changes in receptor and G protein levels were correlated with basic parameters of cardiac function and inotropic responsiveness to isoproterenol. All results were compared with those obtained in age-matched controls. Hypothyroidism in immature rats was associated with markedly reduced myocardial beta-adrenoceptor density, lower content of the long isoform (Gsalpha-L) of the stimulatory G protein and increased amounts of Gialpha2 and Gialpha3. These changes were accompanied by substantially diminished sensitivity to the inotropic effect of isoproterenol. On the other hand, no change in beta-adrenoceptor number, an increased level of Gsalpha-L and decreased levels of Gialpha2 were found in hyperthyroid myocardium. Cardiac inotropic responsiveness to isoproterenol in immature hyperthyroid rats was not significantly altered. In adult hearts of previously hyper- or hypothyroid rats, beta-adrenoceptor density was decreased but G protein levels as well as functional response were comparable to those determined in control animals. It may be concluded that physiological level of thyroid hormones is an important modulator of the normal maturation of the beta-adrenergic system in the developing rat ventricular myocardium. In adult rats previously affected by altered thyroid status, the function of their myocardial beta-adrenergic signaling appears to be compensated despite a lower number of the receptors.

The decrease in the short variant of gsalpha protein is associated with an increase in [3H]CGP12177 binding, [3H]ouabain binding and Na, K-ATPase activity in brown adipose tissue plasma membranes of cold-acclimated hamsters.

Sucrose density gradient purified plasma membranes isolated from brown adipose tissue of cold-acclimated hamsters (4-10 weeks at 0-4 degreesC) were analysed for the content of the short (GsalphaS) and long (GsalphaL) variants of Gsalpha protein (the alpha subunit of the stimulatory G protein) and compared with the membranes isolated from control animals. The relative ratio between the two variants (GsalphaS/GsalphaL) decreased from 0.48 to 0.24 (P<0.01). This result, obtained by electrophoretic resolution of membrane proteins by standard SDS-PAGE and an immunoblot analysis with an antiserum oriented against an internal sequence of Gsalpha, was verified by resolution on urea-containing gels and an antiserum oriented against the C-terminus decapeptide of Gsalpha. Under these conditions, the GsalphaS/GsalphaL ratio was decreased from 0.41 to 0.31 (P<0.05). The total amount of both isoforms (GsalphaS plus GsalphaL) decreased to 83% (P<0.05) or 68% (P<0.01) by standard or urea SDS-PAGE respectively. These data demonstrate that cold-acclimation of hamster brown adipose tissue is associated with preferential decrease in the plasma membrane density of the short variant of the Gsalpha protein.%This decrease was paralleled by an increase in the other plasma membrane constituents, [3H]CGP12177 binding sites, [3H]ouabain binding sites and Na,K-ATPase activity to 147%, 212% and 191% respectively.