The complex regulation of receptor-coupled G-proteins.

Heterotrimeric G-proteins are associated with the cytoplasmic surface of the cell membrane as oligomeric structures. The oligomeric structures were deduced from a variety of studies including target (irradiation) analysis, hydrodynamic evaluation of detergent extracted material, and cross-linking of G-proteins in their membrane environment. From the functional mass determined by target analysis, it was estimated that one receptor (for glucagon) is associated with 8-10 units of Gs, the heterotrimeric G-protein that stimulates adenylyl cyclase.

G proteins: out of the cytoskeletal closet.

This article contains a brief review of GTP-binding proteins (G protein) signaling mechanism with emphasis on accumulated information which suggests that G proteins are multimeric proteins structured such that one receptor can catalytically activate each of the monomers as it moves in an oscillatory fashion along the multimeric chain. Movement is dictated by the binding of GTP or GDP controlled by the exchange reaction induced by agonist binding to the receptor. Based on the dynamic instability model for the interactions of myosin and F-actin, the hypothesis is presented that a GTP-bound monomer is released from one end of the multimer allowing it to interact with effectors such as adenylyl cyclase embedded in the plasma membrane.

The disaggregation theory of signal transduction revisited: further evidence that G proteins are multimeric and disaggregate to monomers when activated.

We have compared the sedimentation rates on sucrose gradients of the heterotrimeric GTP-binding regulatory (G) proteins Gs, G(o), Gi, and Gq extracted from rat brain synaptoneurosomes with Lubrol and digitonin. The individual alpha and beta subunits were monitored with specific antisera. In all cases, both subunits cosedimented, indicating that the subunits are likely complexed as heterotrimers.

Heterotrimeric G proteins in synaptoneurosome membranes are crosslinked by p-phenylenedimaleimide, yielding structures comparable in size to crosslinked tubulin and F-actin.

We have treated rat brain synaptoneurosomes with the crosslinking agent N,N'-1,4-phenylenedimaleimide under conditions that cause extensive crosslinking of tubulin, F-actin, and the alpha and beta subunits of three major types of heterotrimeric GTP-binding regulatory proteins (G(o), Gs, Gi) present in brain membranes. The major crosslinked products are coeluted from Bio-Gel sizing columns as very large structures that do not penetrate stacking gels during SDS/PAGE. The alpha subunits but not the beta subunits of Gs, G(o) and Gi also yield crosslinked products of intermediate sizes.

Glucagon induces disaggregation of polymer-like structures of the alpha subunit of the stimulatory G protein in liver membranes.

The hydrodynamic behavior of G alpha s, the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein (G protein), in octyl glucoside extracts of rat liver membranes was investigated. As was previously shown for G proteins similarly extracted from brain synaptoneurosomes, G alpha s behaved as polydisperse structures with S values higher than that of heterotrimeric G proteins. At concentrations of guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) greater than 100 microM, incubation with membranes led to smaller structures having S values in the range of 4-5 S.

Carbachol-activated muscarinic (M1 and M3) receptors transfected into Chinese hamster ovary cells inhibit trafficking of endosomes.

We examined the effects of isoproterenol and carbachol on fluid-phase endocytosis by Chinese hamster ovary (CHO) cells transfected with beta-adrenergic, M1, or M3 cholinergic receptors. Isoproterenol increased cAMP production and carbachol increased intracellular Ca, indicating successful expression of the receptor genes and coupling to typical signal transduction pathways. Carbachol inhibited the uptake of horseradish peroxidase (HRP) or Lucifer yellow (markers of fluid-phase endocytosis) in both M1- and M3-containing cells but not in wild-type cells, whereas isoproterenol did not affect pinocytosis in cells transfected with beta-adrenergic receptors.

Octyl glucoside extracts GTP-binding regulatory proteins from rat brain "synaptoneurosomes" as large, polydisperse structures devoid of beta gamma complexes and sensitive to disaggregation by guanine nucleotides.

GTP-binding regulatory proteins are generally purified from cholate-extracted membranes in the form of heterotrimers (G proteins) consisting of a GTP-binding subunit (alpha protein) complexed with a tightly interacted heterodimer termed beta gamma. In this study we extracted the proteins from rat brain "synaptoneurosomes" using the neutral detergent 1-octyl beta-D-glucopyranoside (octyl glucoside). Using specific antibodies for detection by immunoblotting and sucrose gradients for analyzing hydrodynamic properties, we found that each species of alpha protein (alpha subunits of stimulatory, inhibitory, and brain GTP-binding proteins) exhibited a broad range (4 S to greater than 12 S) of polydisperse structures with peak values (5 S to 7 S) considerably greater than that of heterotrimeric G proteins.

Microsomal and cytosolic fractions of guinea pig hepatocytes contain 100-kilodalton GTP-binding proteins reactive with antisera against alpha subunits of stimulatory and inhibitory heterotrimeric GTP-binding proteins.

Guinea pig hepatocytes fractionated by differential centrifugation into plasma membrane-enriched, microsomal, and cytosolic fractions were examined for their content of alpha and beta subunits of heterotrimeric GTP-binding proteins (G proteins) involved in signal transduction. alpha subunits of stimulatory (Gs) and inhibitory (Gi) proteins were detected by immunoblots with antisera reactive with the carboxyl-terminal decapeptide regions of these proteins. Unexpectedly, antisera (including immunopurified) to the alpha subunit but not the beta subunit reacted with a band of 100-kDa proteins in both the microsomal and cytosolic fractions.

Isoproterenol stimulates shift of G proteins from plasma membrane to pinocytotic vesicles in rat adipocytes: a possible means of signal dissemination.

Guanine nucleotide-binding regulatory proteins (G proteins) are linked to a large number of surface membrane receptors and appear to regulate a variety of effector systems located both in the plasma membrane and in other parts of the cell. The mechanism of the disseminative actions of G proteins remains obscure. During an investigation of the fate of two types of G proteins, Gs and Gi, in rat adipocytes, we unexpectedly found that isoproterenol, which stimulates cAMP levels and lipolysis in these cells, induces parallel increases in both Gs and Gi in a low-density microsomal fraction rich in endosomes and Golgi bodies.

Pertussis toxin induces structural changes in G alpha proteins independently of ADP-ribosylation.

Pertussis toxin catalyzes ADP-ribosylation of a family of GTP-binding proteins (G alpha proteins) involved in signal transduction. It is thought that this activity is responsible for the attenuating effects of the toxin on the actions of a number of hormones and neurotransmitters. By utilizing specific antisera for detecting on electrophoretic transfer blots (Western blots) alpha proteins that are subject to ADP-ribosylation, it was found that treatment of these proteins with pertussis toxin resulted in shifts in their electrophoretic mobility and marked enhancement of their immunoreactivity compared to untreated proteins.

Photoaffinity labeling of the glucagon receptor with a new glucagon analog.

The preparation, purification and characterization of N epsilon-4- azidophenylamidinoglucagon are described. This photoreactive peptide was found to be 50% as potent as native glucagon in competing with 125I-labeled glucagon for binding to glucagon receptors on rat liver plasma membranes. Similarly, the analog was 50% as potent as native glucagon in its ability to stimulate adenylate cyclase.

Identification and characterization of the rat adipocyte glucose transporter by photoaffinity crosslinking.

The photoaffinity crosslinking agent hydroxysuccinimidyl-4-azidobenzoate has been used to attach [3H]cytochalasin B to a rat adipocyte low-density microsomal membrane protein of 45-50 kDa. The characteristics of the [3H]cytochalasin B-labeled protein are consistent with those of the adipocyte glucose transporter. The low-density microsomes from cells incubated without insulin incorporate twice the amount of radioactivity per mg membrane protein than low-density microsomes derived from insulin-stimulated cells.

Proposed mechanism of insulin-resistant glucose transport in the isolated guinea pig adipocyte. Small intracellular pool of glucose transporters.

A marked resistance to the stimulatory action of insulin on glucose metabolism has previously been shown in guinea pig, compared to rat, adipose tissue and isolated adipocytes. The mechanism of insulin resistance in isolated guinea pig adipocytes has, therefore, been examined by measuring 125I-insulin binding, the stimulatory effect of insulin on 3-0-methylglucose transport and on lipogenesis from [3-3H]glucose, the inhibitory effect of insulin on glucagon-stimulated glycerol release, and the translocation of glucose transporters in response to insulin. The translocation of glucose transporters was assessed by measuring the distribution of specific D-glucose-inhibitable [3H]cytochalasin B binding sites among the plasma, and high and low density microsomal membrane fractions prepared by differential centrifugation from basal and insulin-stimulated cells.

Opiate receptor-mediated inhibition of adenylate cyclase in rat striatal plasma membranes.

Plasma membranes from rat striatum contain adenylate cyclase activity that is subject to dual regulation by GTP. Low concentrations (up to 30 nM) of the nucleotide increase activity whereas higher concentrations evoke a steady decline in activity; such behavior characterizes dually regulated adenylate cyclase systems. The opiates, morphine sulfate and D-Ala-Met-enkephalin, produce naloxone-reversible inhibition of the enzyme that is dependent on "inhibitory concentrations" of GTP (above 50 nM).

Structure-function relationships in adenylate cyclase systems.

Hormone-sensitive adenylate cyclase systems are composed of hormone-recognition units (R), a nucleotide-regulatory unit (N) for reaction with GTP and divalent cations, and the catalytic unit (C). From the reported sizes of purified R and N subunits and target analysis of functional sizes of these units, the functions of the components for the binding and actions of hormones and GTP require minimally dimers, homologous or heterologous. It is proposed that the catalytic unit exists in the membrane also as a dimer and that its transition to the active state with MgATP as substrate involves corresponding transitions in linked dimers of the hormone-recognition and nucleotide-regulatory units.

Structure of the turkey erythrocyte adenylate cyclase system.

Target analysis of the turkey erythrocyte adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.