John D. Hildebrandt

ADP-ribosylation of membrane components by pertussis and cholera toxin.

Publisher Summary Pertussis and cholera toxins are important tools to investigate functional and structural aspects of the stimulatory (Ns) and inhibitory (Ni) regulatory components of adenylyl cyclise. Cholera toxin acts on Ns by ADP-ribosylating its αs subunit. It uses NAD+ as a cosubstrate. ADP-ribosylation of Ns alters its properties so that its guanosine triphosphate (GTP) hydrolyzing capacity is inhibited and the action of GTP is potentiated. In intact cells, this leads to the increases in cyclic adenosine monophosphate (AMP) levels. Both pertussis and cholera toxin are hexameric multisubunit molecules. Cholera toxin is composed of one A and 5 B subunits; pertussis toxin is formed of one S1, one S2, one S3, two S4, and one S5 subunits. This chapter presents a set of protocols, as developed in the laboratory, that can be used to study simultaneously and comparatively the susceptibility of Ns and Ni to be ADP-ribosylated by cholera and pertussis toxin.

Regulation of Hormone Receptors and Adenylyl Cyclases by Guanine Nucleotide Binding N Proteins

Publisher Summary Receptors that affect cyclic adenosine monophosphate (cAMP) are sub-classified into two subtypes: Rs receptors, which increase cAMP levels by stimulating the enzyme adenylyl cyclase, and Ri receptors, which decrease cAMP levels by inhibiting the cAMP-forming enzyme. This chapter discusses the transduction mechanism to which Rs- and Ri-type receptors couple to modulate adenylyl cyclase activity. At the center of this transduction mechanism are two oligomeric coupling proteins called N or G proteins. These proteins have properties to bind and hydrolyze guanosine triphosphate and regulate hormone affinity for receptors and the catalytic activity of the cAMP-forming enzyme. This complex receptor-coupling protein-adenylyl cyclase system is approached by first reviewing structural and functional aspects that regulate cAMP formation. The chapter also discusses the basic structure and regulation of adenylyl cyclase by nucleotides and magnesium. It also discusses action of hormones on the nucleotide-regulated system. It analyzes the known regulation of hormone-receptor interaction by the coupling proteins. The analysis of affinity regulation of receptors leads to conclusions that point toward the existence of at least two conformational states of receptors interacting with at least three conformational states or forms of the coupling proteins.

Role of the Chaperonin CCT/TRiC Complex in G Protein βγ-Dimer Assembly

Gβγ dimer formation occurs early in the assembly of heterotrimeric G proteins. On nondenaturing (native) gels, in vitro translated, 35S-labeled Gγ subunits traveled primarily according to their pI and apparently were not associated with other proteins. In contrast, in vitro translated, 35S-labeled Gβ subunits traveled at a high apparent molecular mass (∼700 kDa) and co-migrated with the chaperonin CCT complex (also called TRiC). Different FLAG-Gβ isoforms coprecipitated CCT/TRiC to a variable extent, and this correlated with the ability of the different Gβ subunits to efficiently form dimers with Gγ. When translated Gγ was added to translated Gβ, a new band of low apparent molecular mass (∼50 kDa) was observed, which was labeled by either 35S-labeled Gβ or Gγ, indicating that it is a dimer. Formation of the Gβγ dimer was ATP-dependent and inhibited by either adenosine 5′-O-(thiotriphosphate) or aluminum fluoride in the presence of Mg2+. This inhibition led to increased association of Gβ with CCT/TRiC. Although Gγ did not bind CCT/TRiC, addition of Gγ to previously synthesized Gβ caused its release from the CCT/TRiC complex. We conclude that the chaperonin CCT/TRiC complex binds to and folds Gβ subunits and that CCT/TRiC mediates Gβγ dimer formation by an ATP-dependent reaction.

γ2 subunit of G protein heterotrimer is an N-end rule ubiquitylation substrate

Heterotrimeric G proteins transduce signals from activated transmembrane G protein-coupled receptors to appropriate downstream effectors within cells. Signaling specificity is achieved in part by the specific α, β, and γ subunits that compose a given heterotrimer. Additional structural and functional diversity in these subunits is generated at the level of posttranslational modification, offering alternate regulatory mechanisms for G protein signaling. Presented here is the identification of a variant of the γ2 subunit of G protein heterotrimer purified from bovine brain and the demonstration that this RDTASIA γ2 variant, containing unique amino acid sequence at its N terminus, is a substrate for ubiquitylation and degradation via the N-end rule pathway. Although N-end-dependent degradation has been shown to have important functions in peptide import, chromosome segregation, angiogenesis, and cardiovascular development, the identification of cellular substrates in mammalian systems has remained elusive. The isolation of RDTASIA γ2 from a native tissue represents identification of a mammalian N-end rule substrate from a physiological source, and elucidates a mechanism for the targeting of G protein γ subunits for ubiquitylation and degradation.

Synthesis and use of biotinylated beta gamma complexes prepared from bovine brain G proteins.

Publisher Summary This chapter describes a method developed for directly studying the association and interaction of the α subunits with the βγ complex. Modified βγ is immobilized on agarose beads, which allows a straightforward binding assay to be performed with α subunits. Biotinylated βγ is prepared by treating intact bovine brain G protein with NHS-biotin, activating with AlF 4 - , and separating the subunits on ω -aminooctyl-agarose column. The b βγ is immobilized on streptavidin–agarose. This allows the association of the various α subtypes with βγ to be studied, and it permits the investigation of factors affecting the interaction of the subunits with one another. Because intact G protein is biotinylated, the binding site(s) on βγ for α is protected from modification. Although the α subunit is heavily biotinylated with this procedure, βγ is minimally modified and appears fully functional. The b βγ is further purified by anion-exchange chromatography. The b βγ is further purified by anion-exchange chromatography. This highly purified, biotinylated βγ appears to maintain all the functional properties of unmodified βγ .

Proteomic Analysis of Bovine Brain G Protein γ Subunit Processing Heterogeneity

We characterized the variable processing of the G protein γ subunit isoforms associated with bovine brain G proteins, a primary mediator of cellular communication. Gγ subunits were isolated from purified brain G proteins and characterized by Edman sequencing, by MALDI MS, by chemical and/or enzymatic fragmentation assayed by MALDI MS, and by MS/MS fragmentation and sequencing. Multiple forms of six different Gγ isoforms were detected. Significant variation in processing was found at both the amino termini and particularly the carboxyl termini of the proteins. All Gγ isoforms contain a carboxyl-terminal CAAX motif for prenylation, carboxyl-terminal proteolysis, and carboxymethylation. Characterization of these proteins indicates significant variability in the normal processing of all of these steps in the prenylation reaction, including a new variation of prenyl processing resulting from cysteinylation of the carboxyl terminus. These results have multiple implications for intracellular signaling mechanisms by G proteins, for the role of prenyl processing variation in cell signaling, and for the site of action and consequences of drugs that target the prenylation modification.

Updated Protocols and Comments on the Purification without Use of Activating Ligands of the Coupling Proteins Ns and Ni of the Hormone Sensitive Adenylyl Cyclase

Ns and Ni have been purified without using NaF and Mg as stabilizing agents (Codina, J., Hildebrandt, J.D., Sekura, R.D., Birnbaumer, M., Bryan, J., Manclark, C.R. and Birnbaumer, L. [1984] J. Biol. Chem. 259, in press). Since the submission of that report, several modifications have been introduced to the purification procedure and additional fractions have been processed from which N proteins are obtained. This article describes the updated protocols and presents methodological details not included in the previous publication. The final products are Ns, the stimulatory N, Ni the inhibitory N, both of subunit structure alpha beta gamma, and a Mr = 40,000 protein of beta gamma composition. They are obtained from human erythrocytes.

Sequence Dependence and Differential Expression of Gγ5 Subunit Isoforms of the Heterotrimeric G Proteins Variably Processed after Prenylation in Mammalian Cells

Between 1 and 2% of proteins coded for in the human genome, including all G protein γ subunits, are predicted to be prenylated. Subsequently, prenylated proteins are proteolytically cleaved at the C terminus and carboxymethylated. These reactions are generally obligatory events required for functional expression of prenylated proteins. The biological role of prenyl substrates has made these reactions significant targets for anticancer drug development. Understanding the enzymology of this pathway will be key to success for this strategy. When Gγ1, -2, -4, -10, -11, -12, and -13 were expressed in HEK293 cells they were completely processed according to the current understanding of the prenylation reaction. In contrast, Gγ5 was processed to two forms; a minor one, fully processed as predicted, and a major one that was prenylated without further processing. When the Ca1a2X motif of Gγ5, CSFL, was exchanged for that of Gγ2, CAIL, Gγ5 was completely processed. Conversely, Gγ2-SFL was incompletely processed. Differential processing of Gγ5 was found due to the presence of an aromatic amino acid in its Ca1a2X motif. Retrieving endogenous Gγ subunits from HEK293 or Neuro-2a cells with FLAG-Gβ constructs identified multiple Gγ subunits by mass spectrometry in either cell, but in both cases the most prominent one was Gγ5 expressed without C-terminal processing after prenylation. This work indicates that post-prenylation reactions can generate multiple products determined by the C-terminal Ca1a2X motif. Within the human genome 10% of predicted prenylated proteins have aromatic amino acids in their Ca1a2X sequence and would likely generate the prenylation pattern described here.

[38] Purification of Ns and Ni, the coupling proteins of hormone-sensitive adenylyl cyclases without intervention of activating regulatory ligands

Publisher Summary This chapter presents a detailed description of a procedure for the purification of N s and N i from human erythrocytes and eliminates any possible alteration of the subunit composition of these proteins as might result from the effect of these ligands to induce their subunit dissociation. The chapter explains that it is now recognized that a large number of hormones and neurotransmitters affect their target cells by modulating cyclic adenosine monophosphate (cAMP) formation in either stimulatory or inhibitory fashion. There are two of these couplings or N proteins—an N s (or G s ), mediating the effects of stimulatory hormone–receptor complexes and a N i (or G i ), mediating the effects of inhibitory or attenuating hormone–receptor complexes. The interaction of a hormone–receptor complex with an N protein then results in an increase in the proportion of the N protein in an active vs. inactive conformation or state, and activated N interacts with the catalyst C of adenylyl cyclase eliciting either an increase in catalytic activity (N s ) or an inhibition of activity (N i ). The chapter also explains that structurally, both N s and N i are αβγ heterotrimers. The activation process of both, N s and N i , is dependent on a guanine nucleotide and Mg and seems to involve not only a conformational change but also a subunit dissociation reaction whereby the αβγ heterotrimers dissociate into an activated α* subunit with guanine nucleotide bound to it (α *G ) and αβγ complex.

Hormonal Regulation of Adenylyl Cyclase Activity

Peptide and protein hormones such as glucagon and gonadotropins and neurotransmitters such as chatecholamines exert their action on target cells by binding to their respective receptors (R). These interactions lead to stimulation of cAMP formation by the adenylyl cyclase systems in these cells. In what follows we shall review and present key experimental evidences on functional aspects of cAMP forma- tion by adenylyl cyclases as seen both in the absence and presence of hormonal influence. We shall present current knowledge on the molecular composition and structure of hormone sensitive adenylyl cyclases. Taking structural as well as functional aspects into account we shall discuss current thoughts on how both hormonal stimulation and the ensuing desensitization to hormonal stimulation come about. Finally we shall present some speculations as to other forms of regulation, especially attenuation of cAMP formation and raise some of the most pertinent questions in signal transduction research.