Alfred G. Gilman

Inhibition of receptor-mediated release of arachidonic acid by pertussis toxin

Treatment of guinea pig neutrophils with pertussis toxin (islet-activating protein; IAP) results in inhibition of N-formyl peptide receptor-mediated release of arachidonic acid and granular enzymes. Inhibition by the toxin is specific, in that responses to the calcium ionophore A23187 are not affected. The action of the toxin is not associated with alterations in cellular concentrations of cyclic AMP but is correlated with the ability of the toxin to catalyze the ADP-ribosylation of a 41,000 dalton membrane protein. This protein comigrates on SDS-polyacrylamide gels with the alpha subunit of Gi, the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. It is likely that this G protein is involved in receptor-mediated signal transduction in neutrophils by mechanisms that do not involve cyclic AMP.

Guanine nucleotide-binding regulatory proteins; mediators of transmembrane signaling

Abstract Binding of a hormone or neurotransmitter to its cell-surface receptor ultimately modulates the activity of one or more membrane-associated intracellular effectors. Members of a family of guanine nucleotide-binding proteins (G proteins) serve as transducers of receptor-generated signals in several such pathways . Michael Graziano and Alfred Gilman review the functions of both G proteins and the effectors that they regulate. Appreciation of an increasing number of these proteins suggests that they coordinate the cells response to a wide variety of functionally diverse signals .

Characteristics of the Guanine Nucleotide-Binding Regulatory Component of Adenylate Cyclase

Publisher Summary This chapter discusses the characteristics of the guanine nucleotide-binding regulatory component of adenylate cyclase. Hormone-sensitive adenylate cyclase is a multiprotein enzyme complex. Its activity is regulated by various hormones, neurotransmitters, and autacoids when the components of the system are properly embedded and organized in the matrix of the plasma membrane. It is demonstrated that a putative guanine nucleotide-binding protein could be partially resolved from the catalytic component of adenylate cyclase by affinity chromatography with GTP-Sepharose. Enzymatic activity that was not adsorbed by the affinity support could only be weakly stimulated by guanine nucleotide analogs; this activity was partially restored by addition of a catalytically inactive fraction that was eluted from the column with a guanine nucleotide. Reconstitution of adenylate cyclase activity in cyc - membranes also provided a method for the quantitative assay of the regulatory protein, G/F. Given this capability and the greater stability of G/F compared to C, purification of the regulatory protein was undertaken.

Role of the β-Adrenergic Receptor in the Regulation of Adenylate Cyclase

Hormones circulating outside of cells influence the metabolic activities of enzymes within them. Although the existence of these interactions has been known, at least in rudimentary form, for close to a century, the identities of the proteins responsible for their mediation are just now being established. Among the many hormonally controlled systems that have been studied, the β-adrenergic receptor—adenylate cyclase complex holds an important place. Studies of it have, in many instances, shaped our ideas about the mechanisms of the signaling process. In part, this has been due to the great variety of chemical compounds that are known to either stimulate or block β-adrenergic receptors. The availability of these compounds has allowed the pharmacological definition of a variety of subclasses of adrenergic receptors. Prominent among them are the α and β receptors and the more recently established distinctions within each of these classes (Langer, 1977).

Guanine-Nucleotide-Binding Regulatory Proteins

Studies of the hormone-sensitive adenylate cyclase complex and of mechanisms of transduction of visual information have led to the discovery of a family of guanine-nucleotide-binding regulatory proteins. This family is now known to include at least four highly homologous members. We can, at this time, formulate general definitions and hypotheses about the family and its functions.

The Guanine Nucleotide-binding Regulatory Component of Adenylate Cyclase

The guanine nucleotide-binding regulatory component of adenylate cyclase (G/F) has been purified from human erythrocyte membranes. It is composed of two major polypeptides with molecular weights of 35,000 and 45,000. When cyc- S49 lymphoma cell plasma membranes are reconstituted with purified human erythrocyte G/F, stimulation of adenylate cyclase by beta-adrenergic agonists, guanine nucleotides, and fluoride is restored. Binding of GTP gamma S to human erythrocyte G/F and GTP gamma S-mediated activation of the protein are closely correlated. The agreement between the apparent dissociation constants for these two reactions suggests that the measured binding site is identical to the site responsible for activation. A 41,000-dalton protein has been identified as a contaminant of preparations of G/F that have been purified by four successive chromatographic steps. This protein serves as a specific substrate for ADP-ribosylation and labeling by islet activating protein (IAP) and [32P]NAD, and it appears to contribute an additional high-affinity guanine nucleotide binding site to such preparations.