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

Abstract

Neurofibromatosis type I (NFI) is a common genetic disorder that causes nervous system tumors, and learning and memory defects in humans, and animal models. We identify a novel growth factor stimulated adenylyl cyclase (AC) pathway 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. We also show that Galpha(s) is the major Galpha isoform in fly brains, and define a second AC pathway stimulated by serotonin and histamine requiring NF1 and Galpha(s), as well as a third, classical Galpha(s)-dependent AC pathway, which 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, we show that Ras activation by hNF1 is essential for growth factor stimulation of AC activity. Further, we demonstrate 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.

Figure 1

NF1 and Ras activate AC. (A) Significant increases in AC activation were observed after 10 min to 60 min incubation with human H-Ras at different concentrations (P < 0.05; n = 3). (B) Both H-Ras and K-Ras stimulate AC activity (1 mm; t = 30 min; n = 4). (C) Rab3a does not stimulate AC activity (1 mm; t = 30 min; n = 4). (D) H-Ras stimulation of AC was eliminated in Nf1^P1 and Nf1^P2 mutant flies, and restored by heat-shock induced expression of a fly Nf1 transgene in hsNfI;Nf1^P2 flies (1 mm; t = 60 min; n = 8, 8, 8, 3). (E) A human NF1–GRD–GST fusion protein is able to stimulate AC, in the absence of H-Ras. There was no stimulation by GST alone, or by NF1–GRD–GST missense mutants, R1391S and K1419Q, that reduce RasGAP activity (1 mm; t = 30 min; n = 4). (F) Stimulation by human NF1–GRD–GST was abolished in Ras^e2F/+ heterozygotes (1 mm; t = 30 min; n = 2). (A–F) Values are mean ± SEM (**P < 0.05; ***P < 0.01).

Figure 2

Growth factors stimulate the novel NF1/Ras-dependent AC pathway. (A) AC activity was significantly increased by treatment of larval brains with 2 mm EGF (n = 18). This stimulation was abolished in EGFR mutants, Egfr^t1, and heterozygotes, Df(2R)Egfr¹⁸/CyO; in Nf1 null mutants, Nf1^P1 and Nf1^P2; and in Ras heterozygotes, Ras^e2F/TM3 and Ras^e1b/TM3 (n = 4). (B) Stimulation of AC by 2 mm TGFα was similarly abolished in the Egfr^t1 mutant, Nf1 mutants and Ras heterozygotes (n = 4). Stimulation of AC by 2 mm EGF (C) or TGFα (D) is not affected in a hypomorphic Gα_s mutant, Gsα^B19, whereas stimulation by 20 mm GTPγS is perturbed (n = 3). (E) There was no stimulation of AC by 2 mm insulin (n = 3). (A–E) Values are mean ± SEM (**P < 0.01; ***P < 0.005).

Figure 3

Neurotransmitters and neuromodulators stimulate two additional AC pathways. FMRFamide and dopamine stimulate Gα_s-dependent AC: activation of AC by 200 nm FMRFamide (A) and dopamine (B) is disrupted in Gα_s mutants, but not in Nf1 mutants or Ras heterozygotes (n = 3–4). Serotonin and histamine however, stimulate NF1/Gα_s-dependent AC: activation of AC by 200 nm serotonin (C) and histamine (D) is disrupted in Gα_s and Nf1 mutants but not in Ras heterozygotes (n = 4). (A–D) Values are mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.005).

Figure 4

Missense mutations and deletions of human NF1 modulate Drosophila MAPK activity. (A) Position of four hNF1 missense mutations, and size of five hNF1 deletion constructs, that have been expressed and analyzed in Drosophila Nf1 null mutants (CSRD, Cys–Ser-rich domain; GRD, GAP-related domain; LRD, Leu-rich domain). Crosses required to generate F1 progeny expressing UAS-hNF1 mutants or deletion constructs under control of the nervous system specific elav-Gal4 driver (B) on the X chromosome or the globally expressing e22c-Gal4 driver (C) on the second chromosome. (D) Representative western blot of head extracts from flies expressing normal and mutant hNF1s and deletions, probed with anti-phospho-MAPK then stripped and re-probed with anti-MAPK antibodies. (E) Levels of phospho-MAPK versus total MAPK levels in flies expressing hNF1 mutants and deletions, normalized to K33 wild-type (+) control values (see Materials and Methods). (D and E) Expression is under control of the e22c-Gal4 driver. (E) Values are mean ± SEM (*P < 0.05; **P < 0.01; n = 4–6).

Figure 5

Separate domains of human NF1 mediate activation of different AC pathways. (A) EGF does not stimulate AC activity in flies expressing RasGAP-defective mutant hNF1s (R1276P, R1391S, K1423E), compared with K33 (control) flies or flies expressing normal hNF1, however, serotonin- and histamine-stimulated AC activity is fully restored. (B) Stimulation of AC activity by EGF, serotonin and histamine is restored in flies expressing the L847P hNF1 mutation. (C) EGF stimulated AC activity is restored in lines expressing GRD fragments (GRD1; GRD2), but serotonin- and histamine-stimulated AC activity is absent. Conversely, serotonin and histamine, but not EGF, stimulate AC activity in flies expressing a GRD deletion (ΔGRD2) or a C-terminal fragment (Cterm) alone. (D) Pupal length is increased in flies expressing normal hNF1 using elav-Gal4 or e22c-Gal4 drivers compared with Nf1 mutant and K33 wild-type (+) controls expressing driver alone. (E) Pupal length is also increased in flies expressing all four missense mutations (L847P, R1276P, R1391S or K1423E) compared with Nf1 mutants expressing driver alone. (F) Pupal length is not increased in flies expressing GRD fragments (GRD1; GRD2) or an N-terminal fragment (Nterm), however it is increased in flies expressing a GRD deletion (ΔGRD2) or a C-terminal fragment (Cterm). (A–C) Expression is under control of the elav-Gal4 driver, values are mean ± SEM (**P < 0.01; ***P < 0.001; n = 4). (D–F) Expression is under control of the e22c-Gal4 driver except where otherwise indicated, values are mean ± SEM (*P < 0.01; **P < 0.001; n > 50).

Figure 6

AC can be activated by at least three distinct pathways: First, a novel NF1/Ras-dependent pathway stimulated by growth factors such as EGF and TGFα that activates an unidentified AC (AC-X), and does not involve Gα_s; secondly, an NF1/Gα_s-dependent pathway, acting through Rutabaga-AC (Rut-AC), stimulated by serotonin and histamine, and possibly PACAP38 (see discussion), that does not require Ras; thirdly, a classical NF1-independent pathway, involving Gα_s but not NF1 or Ras, stimulated by FMRFamide and dopamine that activates an unidentified AC (AC-Y).