Dario Valenzuela

Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP-43.

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. GAP‐43 is a neuronal protein that increases guanine nucleotide exchange by heterotrimeric G proteins. Two cysteine residues near the N‐terminus of GAP‐43 are subject to palmitoylation, and are necessary for membrane binding as well as for G(o) activation. N‐terminal peptides, which include these cysteines, stimulate G(o). Monopalmitoylation reduces, and dipalmitoylation abolishes the activity of the peptides. The activity of GAP‐43 protein purified from brain also is reversibly blocked by palmitoylation. This suggests that palmitoylation controls a cycle of GAP‐43 between an acylated, membrane‐bound reservoir of inactive GAP‐43, and a depalmitoylated, active pool of protein.

Growth cone transduction: Go and GAP-43

Summary The neuronal growth cone plays a crucial role in forming the complex brain architecture achieved during development, and similar nerve terminal mechanisms may operate to modify synaptic structure during adulthood. The growth cone leads the elongating axon towards appropriate synaptic targets by altering motility in response to a variety of extracellular signals. Independently of extrinsic clues, neurons mainfest intrinsic control of their growth and form (Banker and Cowan, 1979). Hence, there must be intracellular proteins which control nerve cell shape, so-called ‘plasticity’ or ‘growth’ genes. GAP-43 may be such a molecule (Skene and Willard, 1981; Benowitz and Lewis, 1983). For example, GAP-43 is localized to the growth cone membrane (Meiri et al. 1986; Skene et al. 1986) and can enhance filopodial formation even in non-neuronal cells (Zuber et al. 1989a). It includes a small region at the amino terminus for membrane association and perhaps growth cone targeting (Zuber et al. 1989b, Liu et al. 1991). We have found that Go, a member of the G protein family that links receptors and second messengers, is the major non-cytoskeletal protein in the growth cone membrane (Strittmatter et al. 1990). Double staining immunohistochemistry for GAP-43 and Go shows that the distributions of the two proteins are quite similar. Purified GAP-43 regulates the activity of purified Go (Strittmatter et al. 1990), a surprising observation since GAP-43 is an intracellular protein. We have compared the mechanism of GAP-43 activation of Go with that of G protein-linked receptors. GAP-43 resembles receptor activation in that both serve primarily to increase the rate of dissociation of bound GDP, with consequent increase in GTPγS binding and GTPase activity. Neither affects the intrinsic rate of hydrolysis of bound GTP by Go. They differ, however, in that pertussis toxin blocks interaction of the receptor with Go, but not that of GAP-43. Furthermore, whereas GAP-43 activates both isolated αo, subunits and αβγ trimers, receptors require the presence of the βγ subunits. Thus like receptors, GAP-43 is a guanine nucleotide release protein, but of a novel class. The interactions between Go and GAP-43 suggest that Go plays a pivotal role in growth cone function, coordinating the effects of both extracellular signals and intracellular growth proteins.

Effect of deletion of the major brain G-protein ? subunit (?o) on coordination of G-protein subunits and on adenylyl cyclase activity

Heterotrimeric G‐proteins, composed of α and βγ 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 α and βγ subunits. Thus far, little is known about how the amounts of α and βγ subunits are coordinated. Targeted disruption of the αo gene leads to loss of both isoforms of αo, the most abundant α subunit in the brain. We demonstrate that loss of αo protein in the brain is accompanied by a reduction of β protein to 32 ± 2% (n = 4) of wild type. Sucrose density gradient experiments show that all of the βγ remaining in the brains of αo−/− mice sediments as a heterotrimer (s20,w = 4.4 S, n = 2), with no detectable free α or βγ subunits. Thus, the level of the remaining βγ subunits matches that of the remaining α subunits. Protein levels of α subunits other than αo are unchanged, suggesting that they are controlled independently. Coordination of βγ to α occurs posttranscriptionally because the mRNA level of the predominant β1 subtype in the brains of αo−/− mice was unchanged. Adenylyl cyclase can be positively or negatively regulated by βγ. Because the level of other α subunits is unchanged and αo itself has little or no effect on adenylyl cyclase, we could examine how a large change in the level of βγ affects this enzyme. Surprisingly, we could not detect any difference in the adenylyl cyclase activity between brain membranes from wild‐type and αo−/− mice. We propose that αo and its associated βγ are sequestered in a distinct pool of membranes that does not contribute to the regulation of adenylyl cyclase. J. Neurosci. Res. 54:263–272, 1998.

Chapter 7: GAP-43 and neuronal remodeling

Publisher Summary This chapter focuses on GAP-43 and presents experiments to determine which components of growth cones distinguish them from synapses. The chapter approaches to determine which nerve terminal components are down-regulated when growth cones become synapses. It identifies several growth cone proteins, one of which was GAP-43, that were diminished by specific target contact in cell culture, suggesting that these proteins played roles more important to growth cones than to synapses. The cDNA for GAP-43 has been cloned to pursue several questions. (1) Which growth cone functions might GAP-43 affect? (2) Is it sufficient by itself to cause any of the structure of the growth cone? (3) How can it be restricted to particular membrane domains of the cells where growth must occur? (4) How is it regulated at the genetic level? (5) Is there a homologous gene in invertebrates? (6) Most importantly, how could an intrinsic GAP-43-induced propensity to remodeling be coordinated with morphological responses induced by the host of known extracellular growth signals?