Ronald Adamik

Protein kinase A-anchoring (AKAP) domains in brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2).

Like other guanine nucleotide-exchange proteins (GEPs) that activate ADP-ribosylation factor (ARF) GTPases, brefeldin A-inhibited GEP2, BIG2, contains an ≈200-aa Sec7 domain that is responsible for this catalytic activity and its inhibition by brefeldin A. The Sec7 domain is located near the center of the molecule and serves to accelerate replacement of GDP bound to ARF with GTP. To explore possible functions of the N-terminal region of BIG2 (1–832), we used three coding-region constructs as bait to screen a human heart cDNA library in a yeast two-hybrid system, retrieving two unique clones that encode a type I protein kinase A (PKA) regulatory subunit, RIα. Coimmunoprecipitation experiments confirmed interaction of in vitro translated BIG2 and RIα, as well as of the endogenous proteins in cytosol of cultured HepG2 cells. Using 28 deletion mutants, we found three regions of BIG2 that interacted with R subunits of PKA. Residues 27–48 (domain A) interacted with RIα and RIβ, 284–301 (domain B) interacted with RIIα and RIIβ, and 517–538 (domain C) interacted with RIα, RIIα, and RIIβ. Sequence analysis and helical wheel projection of amino acids in the three domains revealed potential amphipathic wheel structures characteristic for binding of PKA R subunits. Western blot analysis of subcellular fractions demonstrated translocation of BIG2 (and BIG1) from cytosol to the Golgi and other membrane structures after incubation of cells with 8-Br-cAMP or forskolin. All findings are consistent with a role for BIG2 as an A kinase-anchoring protein (or AKAP) that could coordinate cAMP and ARF regulatory pathways.

Differences in requirements and actions of various histamine-releasing agents

Abstract Histamine release induced by dextran, concanavalin A (con A), antigen, anti-IgE, the ionophore A23187, somatostatin, compound 48/80, polylysine, and protamine was studied in Sprague-Dawley rat mast cells. Release by dextran, con A, antigen, and anti-IgE was highly dependent on addition of phosphatidyl serine (PS). The same agents plus A23187 were highly dependent on exogenous Ca2+ , but only dextran was completely dependent. The other (basic) agents showed little or no dependence on exogenous PS or Ca2+. Addition of 0.1 mM Mn2+ with the A23187 (in the presence of 1.0 mM Ca2+), which probably markedly reduced the level of the A23187-Ca complex, did not greatly educe release by the A23187. Treatment of the cells with neuraminidase increased release by con A, A23187, and antigen; did not greatly affect release by dextran; and definitely decreased release by 48/80, polylysine, and protamine. Each agent desensitized the cells to a higher concentration of the same agent, and most also reduced release by other agents. A23187 and protamine sometimes sensitized to each other, however, and dextran and antigen regularly sensitized to A23187. The observations indicate that the different agents did not act in an identical manner.

Brefeldin A inhibited activity of the sec7 domain of p200, a mammalian guanine nucleotide-exchange protein for ADP-ribosylation factors.

A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cytosol. Cloning and expression of the cDNA confirmed that the recombinant protein (p200) is a BFA-sensitive ARF GEP. p200 contains a domain that is 50% identical in amino acid sequence to a region in yeast Sec7, termed the Sec7 domain. Sec7 domains have been identified also in other proteins with ARF GEP activity, some of which are not inhibited by BFA. To identify structural elements that influence GEP activity and its BFA sensitivity, several truncated mutants of p200 were made. Deletion of sequence C-terminal to the Sec7 domain did not affect GEP activity. A protein lacking 594 amino acids at the N terminus, as well as sequence following the Sec7 domain, also had high activity. The mutant lacking 630 N-terminal amino acids was, however, only 1% as active, as was the Sec7 domain itself (mutant lacking 697 N-terminal residues). It appears that the Sec7 domain of p200 contains the catalytic site but additional sequence (perhaps especially that between positions 595 and 630) modifies activity dramatically. Myristoylated recombinant ARFs were better than non-myristoylated as substrates; ARFs 1 and 3 were better than ARF5, and no activity was detected with ARF6. Physical interaction of the Sec7 domain with an ARF1 mutant was demonstrated, but it was much weaker than that of the cytohesin-1 Sec7 domain with the same ARF protein. Effects of BFA on p200 and all mutants with high activity were similar with ∼50% inhibition at ≤50 μm. The inactive BFA analogue B36 did not inhibit the Sec7 domain or p200. Thus, the Sec7 domain of p200, like that of Sec7 itself (Sata, M., Donaldson, J. G., Moss, J., and Vaughan, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4204–4208), plays a role in BFA inhibition as well as in GEP activity, although the latter is markedly modified by other structural elements.

Interaction of FK506-binding protein 13 with brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1): Effects of FK506

BIG1 and BIG2 are brefeldin A-inhibited guanine nucleotide-exchange proteins that activate ADP-ribosylation factors (ARFs), critical components of vesicular trafficking pathways. These proteins can exist in macromolecular complexes and move between Golgi membranes and cytosol. In the BIG1 molecule, a centrally located Sec7 domain is responsible for ARF activation, but functions of other regions are largely unknown. Yeast two-hybrid screens of a human placenta cDNA library with BIG1 cDNA constructs revealed specific interaction of the N-terminal region (amino acids 1–331) with FK506-binding protein 13 (FKBP13). The association was confirmed by immunoprecipitation of both endogenous BIG1 and FKBP13 from Jurkat T cells with antibodies against either one. Binding of BIG1, BIG2, and ARF to cell membranes in vitro was increased by guanosine 5′-[γ-thio]triphosphate, and further increases were induced by FK506. Incubation of Jurkat T cells with FK506 increased binding of BIG1, BIG2, and ARF to Golgi and other membranes in a time- and concentration-dependent manner, without effects on clathrin or γ-adaptin binding. Binding of BIG1, BIG2, and ARF to membranes was also increased by L-732,531, an agonist structurally related to FK506, but was not increased by a related antagonist, L-685,818, nor by cyclosporin A or rapamycin. These findings are consistent with a role for FKBP13 and FK506 in vesicular trafficking, influencing ARF activity through their guanine nucleotide-exchange proteins.

Reversible inactivation of soluble liver guanylate cyclase by disulfides

Summary Guanylate cyclase activity was reduced in soluble fractions from rat liver slices incubated with diamide, N-ethylmaleimide, or 5′, 5′-dithiobis-2-nitrobenzoate; addition of GSH or dithiothreitol to assays restored activity. Inactivation of purified rat liver guanylate cyclase by p -hydroxymercuribenzoate at 0°C was completely reversed by dithiothreitol. p -Hydroxymercuribenzoate and GSSG caused reversible inactivation of calf liver guanylate cyclase. Dithiobisnitrobenzoate and cystamine were more potent than GSSG; all were more effective at 30°C than at 0°C. It appears that guanylate cyclase activity in intact cells could be modulated by reversible modification of critical sulfhydryl groups, e.g., by thiol:disulfide exchange involving glutathione.

Structural Elements of ADP-ribosylation Factor 1 Required for Functional Interaction with Cytohesin-1

ADP-ribosylation factor 1 (ARF1) is a 20-kDa guanine nucleotide-binding protein involved in vesicular trafficking. Conversion of inactive ARF-GDP to active ARF-GTP is catalyzed by guanine nucleotide exchange proteins such as cytohesin-1. Cytohesin-1 and its Sec7 domain (C-1Sec7) exhibit guanine nucleotide exchange protein activity with ARF1 but not ARF-like protein 1 (ARL1), which is 57% identical in amino acid sequence. With chimeric proteins composed of ARF1 (F) and ARL1 (L) sequences we identified three structural elements responsible for this specificity. Cytohesin-1 increased [35S]guanosine 5′-(γ-thio)triphosphate binding to L28/F (first 28 residues of L, remainder F) and to a much lesser extent F139/L, and mut13F139/L (F139/L with random sequence in the first 13 positions) but not Δ13ARF1 that lacks the first 13 amino acids; therefore, a nonspecific ARF N terminus was required for cytohesin-1 action. The N terminus was not, however, required for that of C-1Sec7. Both C-1Sec7 and cytohesin-1 effectively released guanosine 5′-(γ-thio)triphosphate from ARF1, but only C-1Sec7 displaced the nonhydrolyzable GTP analog bound to mut13F139/L, again indicating that structure in addition to the Sec7 domain is involved in cytohesin-1 interaction. Some element(s) of the C-terminal region is also involved, because replacement of the last 42 amino acids with ARL sequence in F139L decreased markedly the interaction with cytohesin-1. Participation of both termini is consistent with the crystallographic structure of ARF in which the two terminal α-helices are in close proximity. ARF1 residues 28–50 are also important in the interaction with cytohesin-1; replacement of Lys-38 with Gln, the corresponding residue in ARL1, abolished the ability to serve as substrate for cytohesin-1 or C-1Sec7. These studies have defined multiple structural elements in ARF1, including switch 1 and the N and C termini, that participate in functional interactions with cytohesin-1 (or its catalytic domain C-1Sec7), which were not apparent from crystallographic analysis.

Effects of nitroprusside and nitroglycerin on cGMP content and PGI2 formation in aorta and vena cava

Nitroprusside (NP) and nitroglycerin (NG) are potent vasodilators that are used clinically on the basis of their abilities to cause relaxation of smooth muscle. In vitro, both agents cause activation of guanylate cyclase, resulting in increased intracellular cGMP. They also have effects on arachidonate metabolism. Despite apparent similarities in their mechanisms of action, the two drugs have different therapeutic applications based in part on differences in their effectiveness on the arterial and venous systems in vivo. To understand better their target tissue preference, slices of aorta and vena cava were incubated with the agents; cGMP and the vasodilatory prostanoid, prostacyclin, were quantified. NP was more effective in increasing the cGMP content of aorta than of vena cava; it was more active than NG in both tissues. Prostaglandin formation by vascular tissue was influenced by the preliminary equilibration period. Under optimal conditions, it appeared that NG enhanced prostacyclin formation in aorta more than did NP. This in vitro model for NP and NG action may be useful in studying the mechanisms of action of these and other vasoactive agents.

Participation of a guanine nucleotide-binding protein cascade in cholera toxin activation of adenylate cyclase

Guanine nucleotide-binding (G) proteins are involved in several transmembrane signaling systems. Choleragen (cholera toxin) activates adenylate cyclase by catalyzing the ADP-ribosylation of Gs alpha, the stimulatory G protein of the cyclase system. This reaction is enhanced by another guanine nucleotide-binding protein termed ADP-ribosylation factor or ARF that was purified from bovine brain membranes [R. A. Kahn and A. G. Gilman, Journal of Biological Chemistry (1986) 261, 7906-7911]. It was recently found that this ARF also increases the NAD:agmatine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase and auto-ADP-ribosylation activities of the toxin. We have purified and characterized two soluble proteins from bovine brain that act in a similar fashion to enhance choleragen activity in each of these reactions. The membrane and soluble factors are all proteins of approximately 19 kDa that require GTP or GTP analogues for activity and are ADP-ribosylated by the toxin. The ARF proteins apparently interact directly with choleragen in a GTP-dependent fashion to increase its catalytic activity and thus are part of a G protein cascade through which the toxin activates adenylate cyclase. The physiological function of the ARF proteins, as well as their possible relationships to the ras oncogene products and/or the family of G proteins that includes Gs alpha, remains to be determined.

Effects of Calcium and Phosphatidyl Serine in Rat Mast Cell Reaction to Dextran

Summary Histamine release from Sprague–Dawley rat mast cells by dextran was completely inhibited by the absence of exogenous Ca2+ (in contrast to release from the same cells by antigen). Also, spontaneous leakage of histamine from the cells increased in the absence of Ca2+, and cell responsiveness was not completely restored by readding Ca2+. We found no effective substitute for Ca2+ in the release reaction. Ca2+ was not maximally effective immediately when added back to Ca-deficient cells, but almost the full effect of diluting Ca2+ in the medium (which decreased release) and of adding PS (which increased release) were very rapidly established, suggesting that both Ca2+ and PS might act (in part) at superficial cell sites. Release from activated cells could be stopped short by adding glucose or by diluting the cell-dextran mixture with normal buffer, as well as by adding EDTA, which deserves further study.

Separation of the 24 kDa substrate for botulinum C3 ADP-ribosyltransferase and the cholera toxin ADP-ribosylation factor

Botulinum C3 ADP-ribosyltransferase modifies a approximately 24 kDa membrane protein believed to bind guanine nucleotides. Cholera toxin ADP-ribosylation factors are approximately 19 kDa GTP-binding proteins that directly activate the toxin. To evaluate a possible relationship between C3 ADP-ribosyltransferase substrate and ADP-ribosylation factor, they were partially purified from bovine brain. ADP-ribosylation factor, but not C3 ADP-ribosyltransferase substrate, stimulated auto-ADP-ribosylation of the choleragen A1 subunit whereas C3 ADP-ribosyltransferase substrate, but not ADP-ribosylation factor, was ADP-ribosylated by C3 ADP-ribosyltransferase. Thus, although both may be GTP-binding proteins, no functional similarity between ADP-ribosylation factor and C3 ADP-ribosyltransferase substrate was found.