rutabaga

Effect of neurofibromatosis type I mutations on a novel pathway for adenylate cyclase activation requiring neurofibromin and Ras

Neurofibromatosis type I (NFI) is a common genetic disorder that causes nervous system tumors, and learning and memory defects in human and in other animal models. A novel growth factor stimulated adenylyl cyclase (AC) pathway has been identified in the Drosophila brain, which is disrupted by mutations in the epidermal growth factor receptor (EGFR), neurofibromin (NF1) and Ras, but not Galpha_s. This is the first demonstration in a metazoan that a receptor tyrosine kinase (RTK) pathway, acting independently of the heterotrimeric G-protein subunit Galpha_s, can activate AC. This study also shows that Galpha_s is the major Galpha isoform in fly brains, and a second AC pathway is defined stimulated by serotonin and histamine requiring NF1 and Galpha_s. A third, classical Galpha_s-dependent AC pathway, is stimulated by Phe-Met-Arg-Phe-amide (FMRFamide) and dopamine. Using mutations and deletions of the human NF1 protein (hNF1) expressed in Nf1 mutant flies, it is shown that Ras activation by hNF1 is essential for growth factor stimulation of AC activity. Further, it is demonstrated that sequences in the C-terminal region of hNF1 are sufficient for NF1/Galpha_s-dependent neurotransmitter stimulated AC activity, and for rescue of body size defects in Nf1 mutant flies (Hannan, 2006).

This study defines three separate pathways for AC activation: (1) a novel pathway for AC activation, downstream of growth factor stimulation of EGFR that requires both Ras and NF1, but not Galpha_s; (2) an NF1/Galpha_s-dependent AC pathway operating through the Rutabaga-AC (Rut-AC) and stimulated by serotonin and histamine, as observed in the larval brain; (3) a classical G-protein coupled receptor-stimulated AC pathway operating through Galpha_s alone. The Rut-AC pathway may also be stimulated by PACAP38 at the larval neuromuscular junction and in adult heads as shown in previous studies. The AC activated by NF1/Ras (AC-X), or Galpha_s (AC-Y), has not yet been identified (Hannan, 2006).

This study shows for the first time that Ras can stimulate AC in an NF1-dependent manner in higher organisms, via an RTK-coupled pathway that is independent of the Galpha_s G-protein. The functionality of human NF1 in the fly system, and the high degree of identity between human and fly NF1 (60%), suggests that similar pathways for AC activation may also operate in mammals. Previous studies failed to detect stimulation of AC by Ras in cultured vertebrate cell lines, and in Xenopus oocytes, however, these cell types may not contain sufficient NF1 to support NF1/Ras-dependent AC activation. This is consistent with the observation that levels of both Ras and NF1 are critical for stimulation of AC activity in adult head membranes. The reported EGF activation of AC in cardiac myocytes and other tissues requires both Galpha_s, and the juxtamembrane domain of the EGFR, which is not present in the Drosophila EGFR (Hannan, 2006).

Experiments with human NF1 mutants show that the GRD domain and the RasGAP activity of NF1 are both necessary and sufficient for growth factor-stimulated NF1/Ras-dependent AC activity. It is also concluded that C-terminal residues downstream of the GRD are critical for both body size regulation and neurotransmitter-stimulated NF1/Galpha_s-dependent AC activity, thus defining for the first time a region outside the GRD that contributes to this pathway. Interestingly, expression of a human NF1 GRD fragment in Nf1-/- astrocytes results in only partial restoration of NF1-mediated increases in cAMP levels in response to PACAP. Thus, regions outside the GRD also seem to be necessary for activation of AC in these mammalian cells (Hannan, 2006).

Thus, NF1, while being a negative regulator of Ras, is also actively involved in stimulation of AC activity. Moreover, it regulates AC activity through at least two different mechanisms, one of which depends on the RasGAP activity of NF1. The multifunctional nature of the NF1 protein illuminates its importance in nervous system development, tumor formation and behavioral plasticity, and may also explain the wide range of clinical manifestations in neurofibromatosis type I (Hannan, 2006).

Targets of Activity

DCO, the catalytic subunit of Protein kinase A, is preferentially expressed in the mushroom bodies. PKA is the target of cAMP, produced through RUT activity. PKA is a heterotetramer, consisting of two subunits of DC0 and two subunits of a regulatory subunit. Binding of cAMP to the regulatory subunits causes them to dissociate from the tetramer, activating PKA enzymatic activity. Mutants for DCO produce homozygous lethality and a 40% decrease in PKA activity in heterozygotes. This decrease has mild effects on learning but no effect on memory. However, the 80% reduction in activity obtained by constructing double mutant heteroallelic viable animals results in a dramatic learning and memory deficit. These results suggest that PKA plays a crucial role in the cAMP cascade in mushroom bodies to mediate learning and memory processes (Skoulakis, 1993).

Phototransduction in Drosophila is mediated by a G-protein-coupled phospholipase C transduction cascade in which each absorbed photon generates a discrete electrical event, the quantum bump. In whole-cell voltage-clamp recordings, cAMP, as well as its nonhydrolyzable and membrane-permeant analogs 8-bromo-cAMP (8-Br-cAMP) and dibutyryl-cAMP, slow down the macroscopic light response by increasing quantum bump latency, without changes in bump amplitude or duration. In contrast, cGMP or 8-Br-cGMP has no effect on light response amplitude or kinetics. None of the cyclic nucleotides activate any channels in the plasma membrane. The effects of cAMP are mimicked by application of the non-specific phosphodiesterase inhibitor IBMX and the adenylyl cyclase activator forskolin; zaprinast, a specific cGMP-phosphodiesterase inhibitor, is ineffective. Bump latency is also increased by targeted expression of either an activated G_salpha subunit, which increases endogenous adenylyl cyclase activity, or an activated catalytic protein kinase A (PKA) subunit. The action of IBMX is blocked by pretreatment with the PKA inhibitor H-89. The effects of cAMP are abolished in mutants of the ninaC gene, suggesting this nonconventional myosin as a possible target for PKA-mediated phosphorylation. Dopamine (10 µM) and octopamine (100 µM) mimic the effects of cAMP. These results indicate the existence of a G-protein-coupled adenylyl cyclase pathway in Drosophila photoreceptors that modulates the phospholipase C-based phototransduction cascade (Chyb, 1999).

Protein Interactions

Studies in Aplysia and Drosophila have suggested that Ca2+/calmodulin-sensitive adenylyl cyclase may act as a site of convergence for the cellular representations of the conditioned stimulus (Ca2+ influx) and unconditioned stimulus (facilitatory transmitter) during elementary associative learning. This hypothesis predicts that the rise in intracellular free Ca2+ concentration produced by spike activity during the conditioned stimulus will cause an increase in the activity of adenylyl cyclase. However, published values for the Ca2+ sensitivity of Ca2+/calmodulin-sensitive adenylyl cyclase in mammals and in Drosophila vary widely. The difficulty in evaluating whether adenylyl cyclase would be activated by physiological elevations in intracellular Ca2+ levels is in part a consequence of the use of Ca2+/EGTA buffers, which are prone to several types of errors. Using a procedure that minimizes these errors, the Ca2+ sensitivity of adenylyl cyclase in membranes from Aplysia, Drosophila, and rat brain has been quantitified with purified species-specific calmodulins. In all three species, adenylyl cyclase is activated by an increase in free Ca2+ concentration in the range caused by spike activity. Ca2+ sensitivity is dependent on both calmodulin concentration and Mg2+ concentration. Mg2+ raises the threshold for adenylyl cyclase activation by Ca2+ but also acts synergistically with Ca2+ to activate maximally adenylyl cyclase (Yovell, 1992).

The modulatory neurotransmitters that trigger biochemical cascades underlying olfactory learning in Drosophila mushroom bodies have remained unknown. To identify molecules that may perform this role, putative biogenic amine receptors were cloned. One new receptor, DAMB, was identified as a dopamine D1 receptor by sequence analysis and pharmacological characterization. DAMB shows highly enriched expression in mushroom bodies, in a pattern coincident with the rutabaga-encoded adenylyl cyclase. The spatial coexpression of DAMB and the cyclase, along with DAMB's capacity to mediate dopamine-induced increases in cAMP make this receptor an attractive candidate for initiating biochemical cascades underlying learning (Han, 1996).

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