GAP-43 has been termed a "growth" or "plasticity" protein because it is expressed at high levels in neuronal growth cones during development and during axonal regeneration. By homologous recombination, we generated mice lacking GAP-43. The mice die in the early postnatal period. GAP-43-deficient retinal axons remain trapped in the chiasm for 6 days, unable to navigate past this midline decision point. Over the subsequent weeks of life, most GAP-43-deficient axons do enter the appropriate tracts, and the adult CNS is grossly normal. There is no evidence for interference with nerve growth rate, and cultured neurons extend neurites and growth cones in a fashion indistinguishable from controls. Thus, the GAP-43 protein is not essential for axonal outgrowth or growth cone formation per se, but is required at certain decision points, such as the optic chiasm. This is compatible with the hypothesis that GAP-43 serves to amplify pathfinding signals from the growth cone.

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...

We recently described a 125 kd membrane glycoprotein in Saccharomyces cerevisiae which is anchored in the lipid bilayer by an inositol‐containing phospholipid. We now find that when S. cerevisiae cells are metabolically labeled with [3H]myoinositol, many glycoproteins become labeled more strongly than the 125 kd protein. Myoinositol is attached to these glycoproteins as part of a phospholipid moiety which resembles glycophospholipid anchors of other organisms. Labeling of proteins with [3H]myoinositol for short times and in secretion mutants blocked at various stages of the secretory pathway shows that these phospholipid moieties can be added to proteins in the endoplasmic reticulum and that these proteins are transported to the Golgi by the regular secretory pathway. sec53, a mutant which cannot produce GDP‐mannose at 37 degrees C, does not incorporate myoinositol or palmitic acid into membrane glycoproteins at this temperature, suggesting that GDP‐mannose is required for the biosynthesis of these phospholipid moieties. All other secretion and glycosylation mutants tested add phospholipid moieties to proteins normally.

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The yeast Succharomyces cerevisiae has been shown to contain a major 125-kDa membrane glycoprotein which is anchored in the lipid bilayer by a glycophosphatidylinositol anchor. This protein was purified to near homogeneity and was used to raise a rabbit antibody. Biosynthesis of the 125-kDa protein was studied by immunoprecipitation of 35S04-labeled material from wild-type cells or a secretion mutant (secl8) in which the vesicular traffic from the endoplasmic reticulum (ER) to the Golgi is blocked. The 125-kDa protein is first made in the ER as a 105-kDa precursor which already contains a glycophosphatidylinositol anchor and which is slowly transformed into the 125-kDa form upon chase ( t l j z z 10-15 min). The 105-kDa precursor can be reduced to an 83-kDa form by the enzymatic removal of N-glycans. The removal of N-glycans from the mature 125-kDa protein yields a 95-kDa species. Thus, removal of the N-glycans does not reduce the ER and mature forms to the same molecular mass, indicating that not only elongation of N-glycans but also another post-translational modification takes place during maturation. Selective tagging of surface proteins by treatment of 'S04-labeled cells with trinitrobenzene sulfonic acid at 0 "C followed by immunoprecipitation of the tagged proteins shows that the 125-kDa protein, but not the 105-kDa precursor, becomes transported to the cell surface. This tagging of cells after various lengths of chase also shows that the surface appearance of the protein is biphasic with about one half of the mature 125-kDa protein remaining intracellular for over 2 h.Glycosylation and/or glycophosphatidylinositol anchor addition is important for the stability of the 125-kDa protein since the protein remains undetectable in sec53, a temperature-sensitive mutant which does not make GDP-mannose at 37°C and does not add glycophosphatidylinositol anchors at 37 "C.Numerous membrane glycoproteins of protozoan and mammalian origin are anchored in the lipid bilayer by a glycophosphatidylinositol (glycoPtdIns) anchor and the potential functional roles of these anchors have been discussed in recent reviews (Low, 1987; Cross, 1987;Ferguson and Williams, 1988;Low and Saltiel, 1988). The complete structural characterization of the glycoPtdIns anchor of the variant surface glycoprotein from Trypanosoma brucei as well as the mammalian Thy-1 glycoprotein has recently been reported Homans et al., 1988). All evidence argues that the anchor is synthesized as a precursor glycolipid (Krakow et al., 1986; Menon et al., 1988;., 1990a, b) which is transferred en bloc onto glycoproteins shortly after their synthesis (Bangs et al., 1985;Ferguson et al., 1986;. The biosynthesis of the precursor glycolipid in crude microsomes of T. brucei has been reported (Masterson et al., 1989).Yeast cells contain a considerable number of glycoproteins which contain similar glycoPtdIns anchors (Conzelmann et al., 1988(Conzelmann et al., , 1990 Fankhauser and Conzelmann, unpublished). Yeast seems therefore to be an attractive organism to identif...

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We compared excitatory synaptic transmission between hippocampal pyramidal cells in dissociated hippocampal cell cultures and in area CA3 of hippocampal slice cultures derived from wild-type mice and mice with a genetic deletion of the presynaptic growth associated protein GAP-43. The basal frequency and amplitude of action potential-dependent and -independent spontaneous excitatory postsynaptic currents were similar in both groups. The probability that any two CA3 pyramidal cells in wild-type or GAP-43 knockout (-/-) slice cultures were synaptically connected was assessed with paired recordings and was not different. Furthermore, unitary synaptic responses were similar in the two genotypes. Bath application of phorbol 12,13-diacetate (0.6-3 microM) elicited a comparable increase in the frequency of miniature excitatory synaptic currents in wild-type and GAP-43 (-/-) cultures. This effect was blocked by the protein kinase C inhibitor, bisindolylmaleimide I (1.2 microM). Finally, 3 microM phorbol 12,13-diacetate potentiated the amplitude of unitary synaptic currents to a comparable extent in wild-type and GAP-43 (-/-) slice cultures. We conclude that GAP-43 is not required for normal excitatory synaptic transmission or the potentiation of presynaptic glutamate release mediated by activation of protein kinase C in the hippocampus.