Microinjection of a 19-kDa guanine nucleotide-binding protein inhibits maturation of Xenopus oocytes

ADP-ribosylation factors (ARFs) are 19-21-kDa proteins purified from bovine brain that bind guanosine 5'-triphosphate (GTP). They exhibit GTP-dependent activity as activators of cholera toxin-catalyzed ADP-ribosylation of the alpha-subunit of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system (Gs alpha). ARF, which interacts directly with the catalytic subunit of cholera toxin, has no known physiologic role. Intracellular microinjection of ARF was employed to investigate the effect of ARF on progesterone- and insulin-stimulated maturation of Xenopus oocytes. Maturation was inhibited by injection of ARF 3-8 h before exposure of oocytes to progesterone or insulin. ARF inhibition was dependent on progesterone concentration but not on insulin concentration. Inhibition was enhanced by concomitant injection of GTP and to a greater extent by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) which, in the absence of ARF, inhibited somewhat at early time points. The demonstration of this effect of ARF on both progesterone- and insulin-stimulated oocyte maturation may provide a clue to the physiologic role of this guanine nucleotide-binding protein.     

Gz, a guanine nucleotide-binding protein with unique biochemical properties

Cloning of a complementary DNA (cDNA) for Gz alpha, a newly appreciated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the beta gamma subunit complex common to other G proteins; another 26-kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant Gz alpha (rGz alpha) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rGz alpha has several properties that distinguish it from other G protein alpha subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min-1), which is reduced greater than 20-fold in the presence of mM concentrations of Mg2+. In addition, the rate of the intrinsic GTPase activity of Gz alpha is extremely slow. The hydrolysis rate (kcat) for rGz alpha at 30 degrees C is 0.05 min-1, or 200-fold slower than that determined for other G protein alpha subunits. rGz alpha can interact with bovine brain beta gamma but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family.     

Activation of a phosphatidylcholine-specific phospholipase C by colony stimulating factor 1 receptor requires tyrosine phosphorylation and a guanine nucleotide-binding protein.

Receptor tyrosine kinases couple to multiple intracellular effector molecules that are crucial for normal cell growth and transformation. Stimulation of membrane phospholipid hydrolysis by receptor tyrosine kinases is one such pathway for generating intracellular second messengers that may be important for mitogenesis. Certain receptor tyrosine kinases tyrosine phosphorylate a phosphoinositide-specific phospholipase C that hydrolyses the membrane phospholipid phosphatidylinositol 4,5-bisphosphate. In contrast, the glycoprotein receptor for colony stimulating factor 1, a transmembrane tyrosine kinase, does not utilize this pathway, but rather stimulates the hydrolysis of phosphatidylcholine. Here we show that eluates of antiphosphotyrosine affinity purified lysates of colony-stimulating factor 1-stimulated cells contain elevated levels of phosphatidylcholine-specific phospholipase C activity. The affinity-purified activity is sensitive to tyrosine-specific T-cell phosphatase, and is detected in the membrane fraction of stimulated cells. Recovery of phospholipase C activity in the antiphosphotyrosine protein fraction is reduced by pertussis toxin pretreatment of cells. The phosphatidylcholine phospholipase C activity in isolated membranes of colony-stimulating factor 1-treated cells was also reduced by pertussis toxin treatment and stimulated by guanosine 5'-3-O-(thio)triphosphate. These results indicate that colony stimulating factor 1 receptor-mediated stimulation of phosphatidylcholine-specific phospholipase C requires tyrosine phosphorylation, and might be affected by a G-protein coupled pathway.     

Activation of cholera toxin and Escherichia coli heat-labile enterotoxins by ADP-ribosylation factors, a family of 20 kDa guanine nucleotide-binding proteins

Cholera toxin and Escherichia coli heat-labile enterotoxins are responsible, in part, for the symptomatology of cholera and traveller's diarrhoea, respectively. Effects of the toxins result from ADP-ribosylation of regulatory guanine nucleotide-binding (G) proteins; the ADP-ribosylated G protein is stabilized in an activated state, resulting in prolonged effects on its target. Toxin-catalysed ADP-ribosylation is stimulated in vitro by a family of guanine nucleotide-binding proteins, c. 20 kDa, termed ADP-ribosylation factors or ARFs. In the presence of GTP, but not GDP or adenine analogues, ARFs serve as allosteric activators of the toxin. The effects are amplified by certain phospholipids and detergents which promote guanine nucleotide binding. Six different mammalian ARF genes have been identified. They encode highly conserved, ubiquitous proteins of 175 to 181 amino acids, containing consensus domains responsible for guanine nucleotide binding. Differences in amino acid sequences are localized near the amino terminus and in the carboxy half of the protein. Although the physiological functions of ARFs have not been precisely defined, their immunological localization to the Golgi is consistent with a role in the regulated orderly movement of newly synthesized proteins from the endoplasmic reticulum, through the Golgi system to their ultimate destination.     

Specific binding of tubulin to a guanine nucleotide-binding inhibitory regulatory protein in adenylate cyclase system, Ni

A protein was isolated from the soluble fraction of rat brain by affinity chromatography with Sepharose to which guanine nucleotide-binding inhibitory regulatory protein in adenylate cyclase system, Ni, was immobilized. The molecular weight of this protein, specifically bound to the Ni-affinity column, was estimated as 54,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis. Alternately prepared tubulin also bound to the Ni-affinity column. The amino acid compositions of these proteins were also identical. It is strongly suggested that this Ni-binding cytosolic protein is tubulin.     

Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19 kDa protein

Cholera toxin causes the devastating diarrheal syndrome characteristic of cholera by catalyzing the ADP-ribosylation of Gs alpha, a GTP-binding regulatory protein, resulting in activation of adenylyl cyclase. ADP-ribosylation of Gs alpha is enhanced by 19 kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors or ARFs. We investigated the effects of agents known to alter toxin-catalyzed activation of adenylyl cyclase on the stimulation of toxin- and toxin subunit-catalyzed ADP-ribosylation of Gs alpha and other substrates by an ADP-ribosylation factor purified from a soluble fraction of bovine brain (sARF II). In the presence of GTP, sARF II enhanced activity of both the toxin catalytic unit and a reduced and alkylated fragment ('A1'), as a result of an increase in substrate affinity with no significant effects on Vmax. Activation of toxin was independent of Gs alpha and was stimulated 4-fold by sodium dodecyl sulfate, but abolished by Triton X-100. sARF II therefore serves as a direct allosteric activator of the A1 protein and may thus amplify the pathological effects of cholera toxin.     

Reactive sulfhydryl groups of alpha 39, a guanine nucleotide-binding protein from brain. Location and function

The guanine nucleotide-binding proteins which mediate hormonal inhibition of adenylate cyclase as well as hormonal regulation of other membrane functions are alpha, beta, and gamma heterotrimers which are structurally homologous to each other. In brain, the predominant guanine nucleotide-binding component is a 39-kDa protein whose physiological role is as yet unknown. We have used N-ethylmaleimide to define functionally important sulfhydryl groups on alpha 39. Three cysteine residues in the molecule are reactive in unliganded alpha 39. Alkylation of two of these is reduced when guanosine 5'-(3'-O-thio)triphosphate (GTP gamma S) is bound. We have isolated and sequenced tryptic peptides containing the three reactive cysteines. The octapeptide containing the GTP gamma S-insensitive cysteine is at a position equivalent to amino acids 106-113 of the transducin alpha subunit (Lochrie, M. A., Hurley, J. B., and Simon, M. I. (1985) Science 228, 96-99). However, the equivalent peptide in transducin does not contain a cysteine residue. Alkylation of this cysteine blocks ADP-ribosylation of cysteine 351 by pertussis toxin. However, alkylation does not prevent association of alpha with the beta X gamma subunits nor does it inhibit GTPase activity. The two GTP gamma S-sensitive cysteines are at positions equivalent to cysteines 139 and 286 of the transducin alpha subunit. Alkylation of these residues inhibits GTPase activity. Neither of these GTP gamma S-sensitive cysteines are in those regions of alpha 39 which are highly homologous to the GTP-binding site of elongation factor Tu (Jurnak, F. (1985) Science 230, 32-36). However, both are present in the brain 41-kDa guanine nucleotide-binding protein and in the two transducins. The conservation of these cysteine residues suggests that they are important for the function of the subunits.     

Physical and immunological characterization of a guanine nucleotide-binding protein purified from bovine cerebral cortex

ADP-ribosylation by pertussis toxin has been used to identify the alpha subunit of Ni, the guanine nucleotide-binding protein which mediates hormone and GTP inhibition of adenylate cyclase. Two proteins have been purified from bovine cerebral cortex which are substrates for ADP-ribosylation by pertussis toxin, a 41-kDa protein (alpha 41) and a 39-kDa protein (alpha 39). The 41-kDa protein is very similar to the subunit of Ni purified from other tissues while the function of the 39-kDa protein is unknown (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229; Sternweis, P. C., and Robishaw, J. D. (1984) J. Biol. Chem. 259, 13806-13813). We now show that the purified alpha 39 protein from bovine brain is a relatively hydrophilic protein which associates with a hydrophobic beta gamma component. The complex can be dissociated by guanosine 5'-(3-O-thio)triphosphate. The alpha 39 component binds guanosine 5'-(3-O-thio)triphosphate with a KD of 27 nM. We have developed polyclonal antibodies to alpha 39 and beta. The antibodies to alpha 39 cross-react weakly with alpha 41 in an immunoblot assay indicating some homology between the two proteins but making it unlikely that alpha 39 is derived from alpha 41. Using the antibodies for quantitation we found that alpha 39 is 0.5% and beta is 0.7% of membrane proteins. While the antibodies cross-react with alpha 39 and beta proteins in many different species, central nervous system tissues always have more immunoreactivity than membranes from peripheral organs. Anti-beta antibody recognizes the beta subunit when it is associated with alpha 39 or alpha 41 and can immunoprecipitate both alpha . beta gamma trimers. The guanine nucleotide-dependent dissociation of the alpha 39 . beta gamma trimer suggests that the complex could inhibit adenylate cyclase by liberating free beta gamma units. The function of alpha 39 may not, however, be exclusively to regulate adenylate cyclase but may include coupling hormone receptors to other effectors. Antibodies specific for alpha 39 and beta will be useful tools in determining the functions of alpha 39 and beta in hormone-responsive cells.     

Factors determining the specificity of signal transduction by guanine nucleotide-binding protein-coupled receptors. II. Preferential coupling of the alpha 2C-adrenergic receptor to the guanine nucleotide-binding protein, Go.

Cell to cell communication by many hormones and neurotransmitters involves three major entities: receptor (R), G-protein (G), and effector molecule (E). Plasticity in this system is conferred by the existence of each entity as isoforms or closely related subtypes that are expressed in a tissue-specific and developmentally regulated manner. Factors that determine signal specificity in this system are poorly understood. Such factors include the relative affinity and stoichiometry of R-G or G-E and the possible colocalization of R-G-E in cellular microdomains. Utilizing the alpha 2-adrenergic receptor (alpha 2-AR) system as a representative subfamily of this class of signal transducers, we determined the relative importance of these factors. By analysis of R-G coupling in mammalian cells cotransfected with alpha 2-AR genes and G alpha cDNA, we demonstrate preferential coupling between an alpha 2-AR subtype and Go. Our data implicate R-G affinity as an important determinant of signal transduction specificity and indicate that a critical level of Go alpha is required for coupling. This report indicates the utility of R-G cotransfection in mammalian cells as a "natural environment model" to characterize events occurring at the R-G and G-E interface.     

Evidence for a functionally active inhibitory guanine nucleotide-binding regulatory protein in the swine ovary

Incubation of particulate fractions of swine granulosa cells or luteal minces with purified pertussis toxin (islet-activating protein) and [32P]-NAD catalyzed the (32P)-ADP ribosylation of a 41,000 dalton membrane protein. ADP-ribosylation was markedly reduced by prior incubation of intact cells with toxin. The functional relevance of this presumptive inhibitory guanine nucleotide-binding protein in pig granulosa cells was indicated by the ability of prior treatment with pertussis toxin to increase cyclic AMP generation and progesterone production significantly in response to follicle stimulating hormone. Prior cellular intoxication also enhanced cyclic AMP production stimulated by luteinizing hormone and choleratoxin, but not basally or after forskolin. These results demonstrate the presence of an inhibitory guanine nucleotide-binding protein in both the follicular (granulosa cell) and luteal compartments of the mammalian ovary, and indicate its functional relevance in cyclic AMP generation and progesterone secretion.     

The guanine nucleotide-binding protein Gs activates a novel calcium transporter in Xenopus oocytes.

Calcium influx is an important aspect of receptor-mediated signal transduction, yet limited information is available regarding the pathways of calcium influx into nonexcitable cells. We show that treatment of oocytes from Xenopus laevis with cholera toxin, a potent activator of the guanine nucleotide-binding protein Gs, specifically stimulates a sustained inward whole cell flux of calcium through a novel membrane transporter. The calcium is distributed into a mobilizable pool. The flux is voltage-independent and is completely and specifically blocked by microinjection of oocytes with an antiserum directed against Gs alpha. The flux is not activated by treatment of the cells with forskolin or 8-bromo-cyclic adenosine monophosphate indicating that the effect of Gs alpha on the transporter occurs independently of adenylylcyclase activation. Transporter activity is insensitive to benzyl amiloride, does not require a sodium gradient, and is not stimulated by external calcium, indicating that it is not a sodium-calcium exchanger. The Gs-activated flux is dramatically potentiated by lanthanum ion and other trivalent cations but not by any of six divalent cations that were tested; all other known calcium channels and exchangers are, in contrast, potently blocked by lanthanum. The divalent cation cadmium inhibited transporter activity in a concentration-dependent manner. This novel calcium transporter may be important for receptor-mediated calcium influx in the oocyte and perhaps other cell types.     

Hydroxylamine-stable covalent linkage of myristic acid in G0 alpha, a guanine nucleotide-binding protein of bovine brain

G0 alpha, a guanine nucleotide-binding protein with a strong homology to the G1 alpha and Gs alpha regulatory proteins of adenylate cyclase, is shown to contain myristic acid. The attachment of myristate to the protein is stable to hydroxylamine treatment, and since the amino-terminal sequence of G0 alpha is typical of proteins with amino-terminal myristate, the inference is strong that G0 alpha is also myristylated at its amino-terminal glycine.     

Development of the inhibitory guanine nucleotide-binding regulatory protein in the rat testis

The functional development of the inhibitory guanine nucleotide-binding regulatory protein (Gi) and anti-diuretic hormone (ADH) activity was investigated in rat testes. Adult (90-day-old), adolescent (40-day-old), prepubertal (23-day-old), and fetal (20.5 days of gestation) testis cells were cultured with 100 ng/ml pertussis toxin for 24 h. The cells were then cultured with human chorionic gonadotropin (hCG), the ADH agonist arginine vasotocin (AVT), or a combination of the two. Testis cells from rats 23, 40, and 90 days of age that were incubated with hCG increased testosterone production when compared with controls. Preincubation of the cells from postnatal rats with pertussis toxin significantly increased hCG-stimulated testosterone secretion when compared to cells preincubated in medium only at all three ages. AVT suppressed hCG-stimulated testosterone secretion, but this suppression was partially reversed in cells from all postnatal ages preincubated with pertussis toxin. Fetal testis cells showed no response to preincubation with pertussis toxin, even when levels were increased to 400 ng/ml or when pertussis toxin treatment was continued throughout the culture period. AVT also had no effect on fetal testis cells. These results indicate that the Gi protein and AVT are not functional in fetal testes but are active from prepubertal stages of development through maturity.     

The role of a guanine nucleotide-binding protein in the activation of rat liver plasma-membrane adenylate cyclase by forskolin.

The effects of the photoreactive GTP analogue GTP-gamma-azidoanilide on rat liver plasma-membrane adenylate cyclase are described. U.v. irradiation in the presence of the analogue abolished activation by any effector or combination of effectors that function via the activatory G protein. Partial protection against this inhibition was given by F- and guanosine 5'-[gamma-thio]triphosphate. It is concluded that GTP-gamma-azidoanilide acts by a light-induced covalent reaction with the G protein. In the dark the effects of the analogue were similar to those of GTP. Irradiation in the presence of GTP-gamma-azidoanilide was found to reduce but not to abolish activation of rat liver plasma membrane adenylate cyclase by forskolin. The activation by forskolin and GTP together were greater than the sum of the individual activations. Forskolin doubled adenylate cyclase activity in the presence of glucagon and guanosine 5'-[beta, gamma-imido]triphosphate, which might be expected to activate to the maximum possible extent via the G protein. It is concluded that there are two components to the forskolin activation, a guanine nucleotide-dependent and a guanine nucleotide-independent component.     

Solubilization of human placental tumor necrosis factor receptors as a complex with a guanine nucleotide-binding protein.

Human placental membranes exhibited high-affinity receptors for tumor necrosis factor (TNF) (Kd = 5.6 x 10(-10) M) with a density of 1.2-1.7 x 10(10) sites/mg protein. The receptors were solubilized from these membranes with 1% Nonidet P-40, and the solubilized receptor was adsorbed to Con A-Sepharose and wheat germ agglutinin agarose columns, indicating that the TNF receptor derived from human placenta contains carbohydrate chains recognized by these lectins. TNF binding activity was eluted from a column of Sephacryl S-300 as a single peak of Mr 300 kDa. The solubilized receptor was further purified by TNF-Sepharose prepared by coupling of TNF to tresyl-activated Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified sample resolved five major bands of Mr 90, 78, 41, 35, and 11 kDa, suggesting that these polypeptides constitute a multimeric complex with a molecular mass of 300 kDa, as observed in gel filtration study. Furthermore, the TNF-Sepharose-bound fraction demonstrated GTP gamma S binding and GTPase activity. Immunoblot analysis showed that the 41- and 35-kDa polypeptides were recognized by antisera against alpha subunits and beta subunit of GTP-binding proteins, respectively. These results suggest that the native TNF receptor couples to a guanine nucleotide-binding protein to form a large complex structure in human placental membranes.     

Physical and functional association of follitropin receptors with cholera toxin-sensitive guanine nucleotide-binding protein

We have previously reported detergent (Triton X-100) solubilization of a follitropin (FSH) receptor-rich fraction from light membranes of bovine testis that responded to exogenous FSH by activation of adenylate cyclase (Dattatreyamurty, B., Schneyer, A., and Reichert, L. E., Jr. (1986) J. Biol. Chem. 261, 13104-13113). Upon gel filtration of the detergent-extract through Sepharose-6B, two fractions were separated. Each specifically bound [3H]guanosine 5'-imidotriphosphate (Gpp(NH)p) and had guaninetriphosphatase (GTPase) activity. Of these, one fraction (6B-Fraction-1) also bound radioiodinated human follitropin (hFSH), indicating a coelution of the nucleotide-binding protein with receptor. The other fraction (6B-Fraction-2) did not contain detectable FSH receptor activity. Several lines of evidence suggest that 6B-Fraction-1 is a complex consisting of FSH receptor and a guanine nucleotide regulatory protein, probably Ns. 1) The GTP-binding and FSH-binding activities of 6B-Fraction-1 were retained by a GTP-affinity column, and their retention by the affinity matrix could be prevented by simultaneous addition of free Gpp(NH)p. 2) When exogenous GTP was added to 6B-Fraction-1, binding of 125I-hFSH was reduced compared to controls lacking exogenous GTP. This effect of GTP was highly specific and noncompetitive, indicating that GTP did not bind to receptor. In addition, the affinity of receptor for FSH was decreased, and the rate and degree of dissociation of bound labeled FSH from receptor were increased in the presence of exogenous GTP, each in concentration-dependent manner. 3) Exposure of 6B-Fraction-1 to higher concentration of Triton X-100 reduced significantly the receptor-associated GTP-binding activity and also rendered the hormone-binding activity insensitive to GTP. 4) Treatment of highly purified testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminated the ability of GTP to modulate the 125I-hFSH binding to receptor. 5) After cholera toxin-induced [32P]ADP-ribosylation of testis membranes, a major peak of radioactivity (presumably Ns) was coeluted with FSH receptor activity from the Sepharose-6B column. These results and the observation that the effect of GTP is noncompetitive at FSH receptor level suggest that FSH binding inhibition and the increased rate of hormone dissociation from receptor were the result of GTP interaction with a guanine nucleotide regulatory protein, probably Ns, which itself was functionally associated with the FSH receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ontogeny of pulmonary neuroendocrine cells which express the alpha-subunit of guanine nucleotide-binding protein Go

 In order to ascertain that alpha-subunit of guanine nucleotide-binding protein Go (Goα)-positive cells in the lung epithelia are pulmonary neuroendocrine cells (PNECs), we carried out an immunohistochemical study in young adult and fetal lungs of rodents and in cultured fetal lung explants. Serial sections showed that Goα-positive cells were immunostained for calcitonin gene-related peptide and serotonin in young adult mouse, rat, and hamster lungs and that these cells are, therefore, PNECs. In the fetal lungs of hamster and mouse, Goα-positive PNECs appeared in the epithelium of the lobar bronchus by gestational day 13 in hamster and by day 15.5 in mouse, and they increased with a proximal-to-distal wave during the late fetal period. Explants of immature lung from the fetal hamster on gestational day 11 were cultured. After 2 days of culture, Goα-positive PNEC clusters appeared in the main and lobar bronchi and many PNEC clusters were seen after 4 days of culture. To determine the functional significance of Go in the development of the fetal lung, pertussis toxin, a Go inhibitor, was added to the medium, and changes in branching morphogenesis and PNEC development were studied. Although branching morphogenesis was not disturbed by pertussis toxin, the toxin treatment induced large PNEC clusters in the cultured lung explant. In summary, we showed that Goα is a neuroendocrine marker for PNECs and that Goα-positive cells appear along with development of PNECs in fetal hamster lung in vivo and in vitro. The functional significance of Go in the development of fetal lung is obscure, but signals mediated through this GTP-binding protein could be related to some functions of PNECs. Accepted:13 January 1999     

Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin

Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins. Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit. Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken. Levels of ARF were higher in brain than in non-neural tissues. In rat brain, on the second postnatal day, amounts of sARF I and II were similar. By the 10th postnatal day and thereafter, sARF II predominated. Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation. Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes. Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes. From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged. Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product. The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation.