G0, a GTP-binding protein that transduces information from transmembrane receptors, has been found to be a major component of the neuronal growth cone membrane. GAP-43, an intracellular growth cone protein closely associated with neuronal growth, stimulates GTP-gamma-S binding to G0. It does so through an amino-terminal domain homologous to G-linked transmembrane receptors. Thus, G0 in the growth cone may be regulated by intracellular as well as extracellular signals.

Heterotrimeric G proteins, composed of G␣ and G␤␥ subunits, transmit signals from cell surface receptors to cellular effector enzymes and ion channels. The G␣ o protein is the most abundant G␣ subtype in the nervous system, but it is also found in the heart. Its function is not completely known, although it is required for regulation of N-type Ca 2؉ channels in GH 3 cells and also interacts with GAP43, a major protein in growth cones, suggesting a role in neuronal pathfinding. To analyze the function of G␣ o , we have generated mice lacking both isoforms of G␣ o by homologous recombination. Surprisingly, the nervous system is grossly intact, despite the fact that G␣ o makes up 0.2-0.5% of brain particulate protein and 10% of the growth cone membrane. The G␣ o ؊͞؊ mice do suffer tremors and occasional seizures, but there is no obvious histologic abnormality in the nervous system. In contrast, G␣ o ؊͞؊ mice have a clear and specific defect in ion channel regulation in the heart. Normal muscarinic regulation of L-type calcium channels in ventricular myocytes is absent in the mutant mice. The L-type calcium channel responds normally to isoproterenol, but there is no evident muscarinic inhibition. Muscarinic regulation of atrial K ؉ channels is normal, as is the electrocardiogram. The levels of other G␣ subunits (G␣ s , G␣ q , and G␣ i ) are unchanged in the hearts of G␣ o ؊͞؊ mice, but the amount of G␤␥ is decreased. Whichever subunit, G␣ o or G␤␥, carries the signal forward, these studies show that muscarinic inhibition of L-type Ca 2؉ channels requires coupling of the muscarinic receptor to G␣ o . Other cardiac G␣ subunits cannot substitute.Heterotrimeric G proteins, composed of G␣ and G␤␥ subunits, transmit signals from cell surface receptors to cellular effector enzymes and ion channels. One type of G␣ subunit, G␣ o , is extremely abundant in the brain, where it was first identified (1, 2), but it is also expressed in heart, pituitary, and pancreas. In addition to G␣ o , both the brain and the heart contain other closely related G␣ subunits (for example, members of the G␣ i group that are, like G␣ o , substrates for ADP ribosylation by pertussis toxin), as well as G␣ s (which stimulates adenylyl cyclase) and G␣ q (which stimulates phospholipase C␤).The exact function of G␣ o in heart and brain is not known. It is an extremely abundant protein in the nervous system, making up 0.2-0.5% of brain particulate protein (3, 4) and 10% of the growth cone membrane (5). In the nervous system, G␣ o has been postulated to play several roles. The ability of G␣ o to bind GTP␥S can be modulated by GAP43 (neuromodulin), an abundant growth cone protein that is important for neuronal pathfinding (5). Potentially, G␣ o could be part of the signaling cascade that regulates neuronal guidance. Its appearance in the mouse central nervous system is consistent with such a role, since it begins to appear as neurons terminally differentiate and increases as they send out processes (6). The G␣ o protein is conserved in Drosophila, w...

The addition of palmitate to cysteine residues enhances the hydrophobicity of proteins, and consequently their membrane association. Here we have investigated whether this type of fatty acylation also regulates protein-protein interactions. GAP43 is a neuronal protein that increases guanine nucleotide exchange by heterotrimeric G proteins. Two cysteine residues near the N-terminus of GAPF43 are subject to pahmitoylation, and are necessary for membrane binding as well as for Go activation. N-terminal peptides, which include these cysteines, stimulate Go. Monopalmitoylation reduces, and dipalmitoylation aboliUshes the activity of the peptides. The activity of GAP43 protein purified from brain also is reversibly blocked by palmitoylation. This suggests that palmitoylation controls a cycle of GAP43 between an acylated, membrane-bound reservoir of inactive GAP43, and a depalmitoylated, active pool of protein.

Four different structural regions of Escherichia coli tRNAfMet have been covalently coupled to E. coli methionyl-tRNA synthetase (MetRS) by using a tRNA derivative carrying a lysine-reactive cross-linker. We have previously shown that this cross-linking occurs at the tRNA binding site of the enzyme and involves reaction of only a small number of the potentially available lysine residues in the protein [Schulman, L. H., Valenzuela, D., & Pelka, H. (1981) Biochemistry 20, 6018-6023; Valenzuela, D., Leon, O., & Schulman, L. H. (1984) Biochem. Biophys. Res. Commun. 119, 677-684]. In this work, four of the cross-linked peptides have been identified. The tRNA-protein cross-linked complex was digested with trypsin, and the peptides attached to the tRNA were separated from the bulk of the tryptic peptides by anion-exchange chromatography. The tRNA-bound peptides were released by cleavage of the disulfide bond of the cross-linker and separated by reverse-phase high-pressure liquid chromatography, yielding five major peaks. Amino acid analysis indicated that four of these peaks contained single peptides. Sequence analysis showed that the peptides were cross-linked to tRNAfMet through lysine residues 402, 439, 465, and 640 in the primary sequence of MetRS. Binding of the tRNA therefore involves interactions with the carboxyl-terminal half of MetRS, while X-ray crystallographic data have shown the ATP binding site to be located in the N-terminal domain of the protein [Zelwer, C., Risler, J. L., & Brunie, S. (1982) J. Mol. Biol. 155, 63-81].(ABSTRACT TRUNCATED AT 250 WORDS)

Replacement of the solvent-exposed residues of the DNA recognition helix of the 434 repressor with the corresponding residues of the P22 repressor generates a hybrid protein, 434R[a3(P22R)J, which binds specifically to P22 operators. We show here that a new DNA-binding specificity is generated by combining 434 and 434R[a3(P22R)] repressor monomers to form a heterodimer. The heterodimer specifically recognizes a chimeric P22/434 operator that lacks two-fold rotational symmetry.The bacteriophage 434 repressor recognizes its operator DNA as shown in part in Fig. 1. The repressor binds as a dimer, inserting two identical a-helices (recognition helices) into successive major grooves along one face of the DNA. Each recognition helix lies in one-half of the operator, and amino acids on the outside surface of the helix make specific contacts with functional groups exposed in the major groove of the DNA (1). The DNA-binding form of the repressor is a dimer, and dimerization is mediated primarily by the carboxyl domain; the recognition helix is found in the amino domain (2). In the protein-DNA complex, the axis of twofold symmetry of the protein is coincident with that of the two-fold symmetric operator (1).A number of proteins, including the repressors of coliphages A and 434 and the Salmonella phage P22 repressor, use a recognition helix to recognize their operator DNA (see ref. 3 for review). In the "helix-swap" experiment of Wharton and Ptashne (4), the solvent-exposed residues of the 434 repressor recognition helix (referred to as a3 since it is the third a-helix in the protein) were bound to its 14-base-pair (bp) operator is shown. Each monomer is shown as two domains, connected by a linker region. The carboxyl-terminal domains, which mediate dimerization, are shown away from the DNA, whereas the amino-terminal domains contact the operator. The conserved helix-turn-helix motif in each monomer is indicated as a pair of cylinders. The recognition helix, which makes specific contacts to residues in the major groove of the DNA, is shown in black.Cloning of Hybrid Operators. Double-stranded synthetic oligonucleotides carrying the hybrid operators flanked by Sal I-compatible ends were cloned into the Sal I site of plasmid pUC18 (6) to generate plasmids pAD15, pAD16, pAD17, pMUT1, and pMUT3. Plasmid pMUT2 was generated by mismatched primer mutagenesis as outlined below. The polylinker region of plasmid pAD16 was recloned into plasmid pEMBL8 + (7) to give pDV50. Preparation of singlestranded pDV50 DNA from Escherichia coli strain RZ1032 (ATCC 39737; ref. 8) and subsequent mutagenesis were as described by Zoller and Smith (9). All operator sequences were verified by plasmid sequencing using the method of Chen and Seeburg (10).Filter Binding. The hybrid operators were excised from the corresponding plasmids as -80-bp EcoRI-HindIII fragments and were 5' end-labeled to high specific activity at either the EcoRI or HindIII end using polynucleotide kinase and tPresent address:

We show here, both in vivo and in vitro, that P22 repressor binds co‐operatively to operator sites separated by an integral number of turns of the DNA helix. We measure this co‐operativity in vivo using an assay in which repression of a promoter requires co‐operative binding of P22 repressors to two separated (non‐adjacent) operator sites. We report the isolation of mutant repressors that have high affinity for single operator sites, but are defective in co‐operative binding. Six different mutants, all bearing single amino acid changes in the carboxyl domain, have been isolated. We purified the two mutants most deficient in co‐operative binding, and found that they bind non‐co‐operatively in vitro to adjacent as well as to non‐adjacent pairs of operator sites.

Heterotrimeric G-proteins, composed of alpha and betagamma subunits, transmit signals from cell-surface receptors to cellular effectors and ion channels. Cellular responses to receptor agonists depend on not only the type and amount of G-protein subunits expressed but also the ratio of alpha and betagamma subunits. Thus far, little is known about how the amounts of alpha and betagamma subunits are coordinated. Targeted disruption of the alpha(o) gene leads to loss of both isoforms of alpha(o), the most abundant alpha subunit in the brain. We demonstrate that loss of alpha(o) protein in the brain is accompanied by a reduction of beta protein to 32+/-2% (n = 4) of wild type. Sucrose density gradient experiments show that all of the betagamma remaining in the brains of alpha(o)-/- mice sediments as a heterotrimer (s20,w = 4.4 S, n = 2), with no detectable free alpha or betagamma subunits. Thus, the level of the remaining betagamma subunits matches that of the remaining alpha subunits. Protein levels of alpha subunits other than alpha(o) are unchanged, suggesting that they are controlled independently. Coordination of betagamma to alpha occurs posttranscriptionally because the mRNA level of the predominant beta1 subtype in the brains of alpha(o)-/- mice was unchanged. Adenylyl cyclase can be positively or negatively regulated by betagamma. Because the level of other alpha subunits is unchanged and alpha(o) itself has little or no effect on adenylyl cyclase, we could examine how a large change in the level of betagamma affects this enzyme. Surprisingly, we could not detect any difference in the adenylyl cyclase activity between brain membranes from wild-type and alpha(o)-/- mice. We propose that alpha(o) and its associated betagamma are sequestered in a distinct pool of membranes that does not contribute to the regulation of adenylyl cyclase.

Protein affinity labeling groups have been attached to single-stranded cytidine residues in four structural regions of tRNAfMet. Modification of the tRNA with an average of one cross-linking group per molecule is achieved with retention of 75% of the original methionine acceptor activity. Incubation of the modified tRNA with methionyl-tRNA synthetase (MetRS) results in covalent coupling of the protein and nucleic acid by reaction of N-hydroxysuccinimide ester groups attached to the tRNA with lysine residues in the enzyme. In the presence of excess MetRS, approximately 30% of the input tRNA can be covalently bound to protein, indicating that lysine residues are appropriately oriented for reaction with cross-linking groups attached to certain sites in the tRNA but not to others. The cross-linking reaction results in loss of aminoacylation activity of MetRS equal to the amount of covalently bound tRNA. Enzyme activity is restored by release of bound tRNA following cleavage of the disulfide bond of the cross-linker with a sulfhydryl reagent. The data indicate that cross-linking occurs at the tRNA binding site of the enzyme. In the presence of excess modified tRNAfMet, a maximum of 1 mol of tRNA is cross-linked per mol of MetRS, in keeping with the known anticooperative tRNA binding properties of the native dimeric synthetase. In addition, the coupling reaction is effectively inhibited by unmodified tRNAfMet, but not by noncognate tRNAs.