Patent Application: US-54233100-A

Abstract:
neurofibromatosis type 1 protein loss is associated with a variety of disorders . this protein is involved in the metabolic pathway to produce cyclic amp via g protein - coupled receptors . loss in the amount or activity of nf1 protein causes loss of cyclic amp formation and therefore , loss of subsequent protein kinase a activation . administration of nf1 protein or an inducible gene that encodes nf1 protein to individuals with diseases associated with loss of functional nf1 protein results in a satisfactory treatment of the disease .

Description:
the neurofibromatosis type 1 ( nf1 ) tumor suppressor gene product is believed to restrict cell proliferation by functioning as a ras - specific gtpase activating protein ( rasgap ). however , upon generating homozygous null mutations in a drosophila nf1 homolog , no evidence of perturbed rasl - mediated signaling was found , even in genetic backgrounds that reveal subtle abnormalities in ras pathway function . loss of nf1 causes mutants to be reduced in size and mutants also exhibit a diminished escape response . the size defect of nf1 mutants is not modified by manipulating ras1 signaling , but is rescued by increased cyclic amp ( camp )- dependent protein kinase a ( pka ) activity . moreover , mutations that reduce pka levels phenocopy nf1 mutations . these observations suggest that nf1 and pka interact in a pathway that controls the overall growth of the organism . the nf1 protein does not act solely to regulate ras , but also functions as an effector mediating signaling important for differentiation . the present study of drosophila nf1 mutants indicates that the activation of rutabaga ( rut )- encoded adenylyl cyclase ( m . s . livingstone et al ., cell 37 : 205 ( 1984 ); l . r . levin et al ., cell 68 : 479 ( 1992 )) through heterotrimeric guanine nucleotide binding protein ( g protein )- coupled receptors is regulated by the nf1 protein . as described herein , a highly conserved drosophila homolog of the human nf1 tumor suppressor gene has been characterized . molecular and genetic analysis of this gene has resulted in four observations that are relevant to the study of human nf1 function . firstly , the drosophila nf1 protein is similar to mammalian neurofibromin over its entire length , suggesting that additional functional domains reside outside the centrally located gap - and ira - related segments . secondly , the reduced size of nf1 deficient flies reflects a non - autonomous requirement for nf1 , suggesting that some human nf1 symptoms may also have non - cell autonomous origins . it should be noted in this respect that a short stature is among the most common symptoms of nf1 , found in approximately 30 % of patients ( s . m . huson , et al ., brain 111 : 1355 - 1381 ( 1988 ); f . mccormick , curr . opin . gen . dev . 5 : 51 - 55 ( 1995 ); a . bernards , biochem . biophys . acta 1242 : 43 - 60 ( 1995 )). thirdly , ras1 - mediated signals downstream of at least two receptor tyrosine kinases are not detectably perturbed by the complete absence of nf1 . the size and behavioral phenotypes of nf1 mutants may thus reflect roles for nf1 in ras1 - mediated pathways downstream of other receptor types , e . g ., g protein - coupled receptors , or even reflect functions for nf1 unrelated to its role as a ras regulator . finally and most intriguingly , the size defect of nf1 mutants is not modified by manipulating ras1 pathway components , but is mimicked by reducing pka levels and rescued by increasing pka activity . if nf1 and pka do indeed function in the same pathway , one possibility is that nf1 regulates pka activity . fig1 a , 1 b and 2 show the drosophila nf1 gene structure and comparison of the encoded protein to human neurofibromin . fig1 a shows the intron - exon structure and location of translational start and stop codons . the location of a p - element in nf1 p2 and the extent of the deletion in nf1 p1 are indicated . fig1 b shows the percentage amino acid sequence identity between the indicated segments of drosophila and human nf1 . the grd and ira - related segments are drawn as black and shaded boxes , respectively . fig2 shows the alignment of drosophila ( dm ) and human ( hs ) nf1 proteins . dashes were introduced to optimize the alignment . amino acids encoded by the last complete codon in each exon are labeled with ⋄ signs . the boxed segment shows the approximate extent of the grd . three positions where alternate splicing inserts short segments in human neurofibromin ( t . nishi , el al ., oncogene 6 : 1555 - 1559 ( 1991 ); d . h . gutmann et al ., hum . mol . genet . 2 : 989 - 992 ( 1993 ); g . danglot , et al ., hum . mol . gen . 4 : 915 - 920 ( 1995 )) are identified by filled - in triangles . one of these locations corresponds exactly to the position where drosophila exon 17 is joined to either exon 18a or 18b . exon 18b includes a translational terminator after a single codon and cdnas harboring this exon predict a protein ending in ptdkaa ( seq id no : 3 ). 11 out of 17 drosophila splice sites map within two codons of splice sites in the human nf1 gene ( y . li , et al . genoinics 25 : 9 - 18 ( 1995 )). the drosophila homolog of nf1 is 60 % identical to the human nf1 protein over its entire 2802 amino acid length . although homozygous loss of nf1 is lethal in mice ( c . i . brannan et al ., genes & amp ; development 8 : 1019 ( 1994 )), two viable drosophila null mutations of nf1 have been generated . there is no nf1 protein detected by protein immunoblotting in these two mutants . nf1 p1 is a small deletion that includes the nf1 locus and at least two adjacent genes whereas nf1 p2 represents a p - element insertion . modulation of voltage - activated k + currents ( y . zhong et al ., neuron 14 : 527 ( 1995 ); y . zhong , nature 375 . 588 ( 1995 )) induced by the neuropcptide pituitary adenylyl cyclase activating polypeptide ( pacap38 ) is eliminated in these two mutant alleles . pacap38 [ which belongs to the vasoactive intestinal polypeptide - secretin - glucagon peptide family and stimulates camp synthesis through g protein - coupled receptors in vertebrates ( a . arimura , regulatory peptides 37 : 287 ( 1992 ); d . spengler et al ., nature 365 : 170 ( 1993 ))] induces a 100 - fold enhancement of k + currents by co - activating both rut - adenylyl cyclase - camp and ras - raf kinase pathways ( y . zhong , nature 375 : 588 ( 1995 )). mutations in the rut ( m . s . livingstone et al ., cell 37 : 205 ( 1984 )), ras ( m . a . simon et al ., cell 67 : 701 ( 1991 )), or raf ( l . ambrosio et al ., nature 342 : 288 ( 1989 )) loci eliminate the response to pacap38 ( y . zhong , nature 375 : 588 ( 1995 )). activation of both camp and ras - raf pathways together , but not alone , mimics the pacap38 response ( y . zhong , 1995 )). the involvement of ras in the pacap38 response led to the investigation of the effect of nf1 mutations described herein . the neurofibromatosis type 1 ( nf1 ) tumor suppressor protein partially functions as a ras - specific guanosine triphosphatase ( gtpase ) activating protein . study of drosophila nf1 mutants reveals that nf1 is essential for the cellular response to the neuropeptide pacap38 ( pituitary adenylyl cyclase - activating polypeptide ) at the neuromuscular junction . the peptide induces a 100 - fold enhancement of k + currents by activating the ras - raf and adenylyl cyclase - adenosine 3 ′- 5 ′ monophosphate ( camp ) pathways . this response was eliminated in nf1 mutants . nf1 appears to regulate the rutabaga - encoded adenylyl cyclase , rather than the ras - raf pathway . moreover , the nf1 defect was rescued by exposure of cells to pharmacological treatment that increased concentrations of camp . in summary , signaling by the pacap38 neuropeptide is impaired in nf1 mutants and the defect is apparently caused by a blockade of pacap38 - stimulated activation of rut - adenylyl cyclase . thus , the nf1 protein not only acts as a negative regulator of ras but also as a crucial component for activation of the camp pathway . the induced expression of a catalytic subunit of camp - dependent protein kinase rescues the developmental phenotype of small body size in nf1 p1 and nf1 p2 mutants , providing further support for the above conclusion . the invention is further illustrated by the following specific examples . these examples should not be construed as limiting the invention in any way . to analyze nf1 function in an organism amenable to genetic analysis , a conserved drosophila nf1 homolog was identified . drosophila nf1 clones were isolated by screening a canton - s λ , fix ii genomic phage library ( stratagene ) in 25 ′ 7 / 0 formamide at 37 ° c . with a probe representing the c - terminal 1598 amino acids of human nf1 . all clones identified represented the same locus . a 13 , 295 bp segment representing the entire gene was sequenced . a set of 32 overlapping cdnas was isolated from eye disc , total disc and mixed stage embryo libraries and used to determine 9750 bp of overlapping cdna sequence . the genomic and cdna sequences differ in multiple locations , but none of these polymorphisms affect the sequence of the encoded protein . the genbank accession numbers for the genomic and alternatively spliced cdna sequences are l26500 , l26501 and l26502 . comparing 13 , 295 bp of genomic and 9750 bp of cdna sequence shows that drosophila nf1 consists of 17 constitutive and 2 alternatively spliced exons 18a and 18b . the alternatively spliced cdnas predict proteins of 2764 and 2802 amino acids that are 60 % identical to the human nf1 protein , neurofibromin ( fig1 and 2 ). sequence similarity is observed over the entire length of the proteins , including regions outside of the catalytic gap - related domain ( grd ) or the more extensive segment related to yeast ira proteins ( g . xu , et al ., cell 62 : 599 - 608 ( 1990 )). no related sequences were identified during low stringency screens of several cdna and genomic libraries , indicating that the identified gene is the only drosophila nf1 homolog . rna in situ hybridization and staining of embryos and imaginal discs with monoclonal antibodies against the drosophila protein indicate that nf1 is widely expressed at low levels during all developmental stages . in situ hybridization with single - stranded digoxygenin labeled probes and antibody staining of whole - mount preparations was performed as described ( n . h . patei , in drosophila melanogaster : practical uses in cell and molecular biology l . s . b . goldstein , e . a . fyrberg , eds . academic press , san diego , 1994 ). monoclonal antibodies against drosophila nf1 were generated , after immunizing mice with an affinity purified his - tagged fusion protein representing the c - terminal 450 residues of the longer isoform . the drosophila nf1 gene was mapped to cytogenetic interval 96f and subsequently localized to a 30 kb dna segment between the bride ofsevenless gene and the enhancer of split [ e ( spl )] complex . the nf1 homolog was mapped to cytogenetic interval 96f by in situ hybridization of biotinylated probes to salivary gland chromosomes ( w . r . engels et al ., focus 8 : 6 - 8 ; ( 1996 ). using available genomic clones from this region , the gene was subsequently sublocalized to the 30 kb interval between the bride of sevenless and e ( spl ) loci . this deficiency uncovers the nf1 locus ( hart , a . c . et al ., genes and devel ., 4 : 1835 - 1847 ( 1990 )). to isolate mutant alleles at the nf1 locus , ‘ local hops ’ from a strain ( k33 ), which harbors a p - element transposon within the e ( spl ) complex , approximately 15 kb downstream of nf1 , were generated . inverse pcr was used to screen . flies were raised on standard medium and crosses carried out at 25 ° c . to generate nf1 mutants , w ; p [ w ] males homozygous for a p [ w ] transposon in 96f were crossed to virgin ki pp δ2 - 3 transposase bearing females . single f t dysgenic males of genotype w ; p [ laczw ]/ kip p δ2 - 3 were crossed to w ; tm3 / tm6b virgin females . single f 2 males of genotype w ; p [ iaczw ]/ tm3 or w , p [ iaczw ]/ tm6b were crossed to w ; tm3 / tm6b virgin females to establish lines with stable novel p - element integrations . the red eyed progeny of this cross was analyzed in pools by inverse pcr ( b . dalby , et al ., genetics 139 : 757 - 766 ; ( 1995 ). among 1600 lines screened , two showed evidence of de novo transposon insertions within the nf1 gene . sequence analysis of cloned insertion sites and detailed mapping showed that in one mutant allele , nf1 p1 , a deletion has removed all of the nf1 gene except for the first exon ( fig1 a ). the deletion extends from the first nf1 intron to the site of the original p - element insertion and as a consequence also removes at least two e ( spl ) transcripts . the other allele , nf1 p2 , contains a p - element in the first nf1 intron ( fig1 a ). both alleles fail to express any detectable nf1 protein and hence represent null mutations at this locus . unlike nf1 - deficient mice ( c . i . brannan , et al . genes dev . 8 : 1019 - 1029 ( 1994 ); t . jacks , et al ., nature genetics 7 : 353 - 361 ( 1994 )), drosophila nf1 mutants are viable and can be maintained as robust homozygous stocks . however , while heterozygotes ( nf1 /+) show no obvious defects , homozygotes ( nf1 / nf1 ) of either allele are 25 - 30 % smaller than the parental k33 strain during all post - embryonic stages . this growth defect is apparent under a variety of culture conditions and mutant animals do not display delayed eclosion or bristle phenotypes that are observed with several minute mutations ( k . kongsuwan et al ., nature 317 : 555 - 558 ( 1985 )). the growth defect is fully rescued by expression of an inducible hsnf1 transgene . a hsp70 - nf1 mini gene was generated by cloning a hybrid cdna / genomic nf1 insert into the sacil and kpn1 sites of the pkb176pl p - element vector ( k . basler et al ., science 243 : 931 - 934 ( 1989 )). the genomic segment of the insert harbors three introns and is flanked by mlui and esp3i sites . this vector was introduced into the germline of w 1118 flies . rescue was obtained with daily , 30 minute 37 ° c . heat shocks . to determine whether reduced cell proliferation or impaired cell growth underlies the smaller size of nf1 mutants , the wings of wild - type and mutant animals were compared . the linear dimensions of nf1 mutant wings are 25 - 30 % smaller than those of wild - type flies . since each wing epidermal cell secretes a single hair , cell densities can be determined by counting the number of hairs in a defined region ( t . dobzhansky , wilhelm roux &# 39 ; archivfuer entwicklungsmechanik der organismen 115 : 363 - 379 ( 1929 )). using this approach , both homozygous nf1 mutants were found to have a 30 - 40 % higher cell density compared to the parental line . thus , at least in the wings , reduced cell size contributes significantly to the reduction in overall dimensions , implicating nf1 in a process which regulates cell growth . to determine whether the reduced size of wing epidermal cells reflects a cell autonomous defect , x - irradiation was used to induce mitotic recombination in the wings of heterozygous nf1 mutants , using bald andforked bristle markers to distinguish homozygous mutant clones from surrounding tissue . clonal analysis was performed in a forked ( ƒ ) background . females of genotype ƒ : bld p [ ƒ +] tm3 ser were crossed to either nf1 p1 , nf1 p2 or k33 males . parents were removed after 24 hrs and the larval progeny x - irradiated ( 1000 rads ) after 48 - 72 hrs . adult f 1 males of genotype ƒ : bld p [ ƒ +]/ nf1 p1 or ƒ ; bld p [ ƒ +]/+ were analyzed . no difference in the distance between wing hairs was observed between multiple nf1 −/ clones and surrounding tissue . the reduced size of wing cells therefore reflects a non - autonomous requirement for nf1 , perhaps reflecting a hormonal deficiency or impaired nutrition or metabolism . however , while smaller cells contribute to the reduced size of wings , the eyes of nf1 mutants show a reduced number of ommatidia of normal size and structure . furthermore , nf1 deficient embryos are of normal size . thus , loss of nf1 affects the growth of different tissues in different ways . since the only known biochemical property of the nf1 protein is its ability to negatively regulate ras ( t . n . basu , et al ., nature 356 : 713 - 715 ( 1992 ); j . e . declue , et al ., cell 69 : 265 - 273 ( 1992 ); h . a . kim et al ., oncogene 11 : 325 - 335 ( 1995 ); g . bollag , et al ., nat . genet . 12 : 144 - 148 ( 1996 ); d . a . largaespada et al ., nat . genet . 12 : 137 - 143 ( 1996 )), it was surprising that nf1 mutants did not exhibit phenotypic abnormalities associated with excess ras 1 or ras 2 activity . indeed , whereas expression of activated ras 1 or ras 2 transgenes results in widespread developmental defects , nf1 mutants are smaller but otherwise patterned normally . the regulation of ras by nf1 both in vitro and in vivo was therefore examined . to confirm that drosophila nf1 can act as a rasgap , gap assays with bacterial fusion proteins representing the catalytic domains of human p 120gap , and human or drosophila nf1 were performed . gap assays were performed as described ( s . brill , et al ., mol . cell . biol . 16 : 4869 - 4878 ( 1996 ), with appropriately diluted lysates of bacteria expressing soluble fusion proteins . lysates were standardized for total protein content and fusion protein expression level . the human nf1 and p120 - gap catalytic domain fusion proteins were as described ( g . a . martin , et al ., cell 63 : 843 - 849 ( 1990 ); g . a . martin et al ., science 255 : 192 - 194 ( 1992 )). the drosophila nf1 catalytic domain ( amino acids 1235 - 1614 ) was cloned into pgex20 . all three fusion proteins stimulated the gtpase activity of h - ras , but not of the constitutively active h - ras va112 mutant . thus , drosophila nf1 clearly functions as a rasgap in vitro . ras 1 function in vivo was then examined . the drosophila ras 1 protein performs a crucial function in signaling pathways downstream of several receptor tyrosine kinases ( rtks ), including torso and sevenless . since minor perturbations in ras 1 function have phenotypic consequences in each of these pathways , loss of nf1 perturbations of torso - controlled specification of embryonic terminal structures or sevenless - mediated photoreceptor differentiation were examined . the pattern of tailless expression , which is regulated by torso ( j . b . dully et al ., dev . biol . 166 : 380 - 95 ( 1994 )), is normal in nf1 deficient embryos . thus , nf1 does not appear to be an essential regulator of torso signaling . to test for abnormalities in sevenless signaling ( m . a . simon et al ., cell 67 : 701 - 716 ( 1991 ); b . dickson et al ., curr . opin . genet . dev . 4 : 64 - 70 ( 1994 ); l . bonfini et al ., science 255 : 603 - 606 ( 1992 )), the retinas of mutant animals were examined . retinas of nf1 p2 homozygotes , of nf1 ′/ nf1 p1 and of nf1 p2 / df ( 3r ) boss 15 ( 17 ), are completely wild type implying that ras1 - mediated determination of retinal cell fates is unperturbed . this deficiency uncovers the nf1 locus ( a . c . hart et al ., genes and devel . 4 : 1835 - 1847 ( 1990 ). in homozygotes of nf1 p1 , 25 % of ommatidia have one or more extra photoreceptor cells ( not shown ). however , this phenotype may be due to deletion of genes within the neurogenic e ( spl ) complex , which has occurred in the nf1 p1 allele . a particularly sensitive indicator of sevenless pathway function is the sevr e4 ; sos jc2 /+ mutant combination . only approximately 17 % of ommatidia in this double mutant have r7 cells , and this number is very sensitive to alterations in the gene dosage of ras1 pathway components ( r . d . rogge et al ., cell 64 : 39 - 48 ( 1991 )). flies of this genotype which are also heterozygous for nf1 p2 had no significant alteration in the fraction of r7 containing ommatidia ( not shown ). thus , at least two ras1 - mediated signaling pathways downstream of rtks were not influenced by a reduction in nf1 function . although nf1 does not appear to be a major regulator of ras1 in rtk - mediated signaling , the nf1 deficient phenotype may still reflect improper regulation of other less well characterized functions of ras1 , e . g ., in signaling downstream of g - protein - coupled receptors . if so , then manipulating the level or activity of ras1 pathway components may modify the nf1 deficient phenotype . loss of function mutants used in this work include : sos e2h , ras1 e1b , ras1 e2f ( m . a . simon et al ., cell 67 : 701 - 716 ( 1991 )), and dco 3b , dƒ ( 2l ) tw2 ( d . kalderon et al ., genes dev . 2 : 1539 - 56 ( 1988 ); m . e . lane et al ., genes dev . 7 : 1229 - 43 ( 1993 )). raf gof is an activated allele of drosophila raf ( a . h . brand et al ., genes dev . 8 : 629 - 639 ( 1994 )). hsp70 - pka * flies harbor a murine pka transgene with his87gln and trp196arg substitutions that block interaction with the pka regulatory subunit ( j . jiang et al ., cell 80 : 563 - 572 ( 1995 )). expression of this gene was induced by daily , 30 minute 37 ° c . heat shocks or by growing cultures at 25 ° c . however , heterozygous loss of ras1 or sos had no effect on the size of nf1 mutant pupae , nor did crossing in an activated raf gof mutation . neither reducing nor increasing signaling through the ras1 - raf pathway therefore modifies the nf1 phenotype . this raised the possibility that the nf1 mutant phenotype may not involve ras - raf mediated signaling . role of the nf1 gene product in the adenylyl cyclase - protein kinase pathway . other workers previously observed that flies carrying a viable heteroallelic combination of mutant alleles of the gene encoding the pka catalytic subunit , dco , were reduced in size ( e . m . skoulakis et al ., neuron 11 : 197 - 208 ( 1993 )). for this reason and because an electrophysiological phenotype of nf1 mutants is rescued by camp , the camp - pka pathway representing an alternate target for nf1 was tested . pupae from a heteroallelic combination of dco mutations ( dco tw2 / dco b3 ) were examined and it was found that these are phenotypically indistinguishable from nf1 mutants . whether increasing pka activity in nf1 mutant animals would rescue the size defect was then tested . this was achieved by expressing a constitutively active murine pka catalytic subunit transgene in an nf1 mutant background . heat shock induced expression of this mutant protein , resulted in lethality . however , lower levels of transgene expression were achieved by growing the cultures at 25 ° c . under these conditions , significant rescue of the pupal size defect was consistently observed . in contrast to its effect on nf1 mutant pupae , the pka transgene did not modify the phenotype of tubby , a mutation that results in pupae of small size . the dominant tubby ( tb ) mutation maps to 3 - 90 . 6 , near nf1 ( d . l . lindsley et al ., the genetics and biology of drosophila melanogaster , academic press , san diego , 1992 ). however , 2 . 9 % genetic recombination observed between tb and the p [ w ] transposon in the k33 strain indicates that tb is not an allele of nf1 . nor were wild - type flies expressing the pka transgene observed to be larger . since expression of activated pka suppresses the phenotype of null alleles of nf1 , pka cannot function upstream of nf1 in a simple linear pathway . therefore , pka must either function downstream of nf1 or in a parallel pathway . in addition to their reduced size , nf1 mutants also display a behavioral defect characterized by a diminished escape response . thus , in an assay that determines the number of flies that escape either spontaneously within 90 seconds of release or after repeated prodding ( s . richards et al ., genetics 142 : 1215 - 1223 ( 1996 )), approximately 15 % of either nf1 mutant ( n = 200 ) failed to respond , as compared to 3 % non - responders for the parental k33 strain . the reduced escape rate does not reflect obvious anatomical defects of the peripheral nervous system or the musculature , and the mutants scored within normal limits in tests measuring their activity or their response to visual or olfactory stimuli ( r . wehner , j insect physiol . 18 : 1531 - 1543 ( 1972 ); p . monte , et al ., behavior genetics 19 : 267 - 283 ( 1989 )). because opening of a post - synaptic k + channel in the larval neuromuscular junction requires both ras1 and adenylyl cyclase ( y . zhong , nature 375 : 588 - 592 ( 1995 )), it seemed possible that aberrant neuromuscular function might explain the diminished escape response . indeed as described further below , a specific electrophysiological defect was demonstrated at the larval neuromuscular junction , which is rescued by pharmacological manipulation of the camp - pka pathway and is insensitive to manipulation of ras1 - mediated signaling . therefore , activation of pka rescues at least two phenotypes associated with loss of nf1 . requirement of drosophila nf1 protein for activation of adenylyl cyclase by pacap38 - like neuropeptides pacap38 - induced responses were recorded by the two - microelectrode voltage - clamp method from body - wall muscle fibers of larvae at the third instar ( y . zhong , nature 375 : 588 ( 1995 ); a . arimura , regulatory peptides 37 : 287 ( 1992 ); d . spengler et al ., nature 365 : 170 ( 1993 ); l . y . jan et al ., j . physiol . 262 : 189 ( 1976 ); c .- f . wu et al ., science 220 : 1076 ( 1983 ); b . a . stewart et al ., j comp . physiol . 175 : 179 ( 1994 )). electrophysiological recording : the larval body - wall neuromuscular preparation has been described ( y . zhong et al ., neuron 14 : 527 ( 1995 ); y . zhong , nature 375 : 588 ( 1995 ); l . y . jan et al ., j physiol . 262 : 189 ( 1976 ); c .- f . wu et al ., science 220 : 1076 ( 1983 ); b . a . stewart et al ., j comp physiol . 175 : 179 ( 1994 )). the setup , saline , recording conditions and voltage paradigms were as described ( y . zhong et al ., neuron 14 : 527 ( 1995 ); y . zhong , nature 375 : 588 ( 1995 )). for recording the pacap38 - induced synaptic current , the membrane potential was clamped at − 80 mv . for recording k + currents , command voltages were stepped from the holding potential of − 80 to − 50 and + 20 mv , respectively . these currents include outward k + and inward ca 2 + currents , but the inward ca 2 + component is completely masked ( s . singh et al ., neuron 2 : 1325 ( 1989 )). pacap38 was applied by pressure ejection through a glass electrode positioned near the voltage - clamped muscle membrane . forskolin and camp analogs were applied to the solution bathing the preparation . perfusion of pacap38 to the neuromuscular junction induced an inward current followed by a 100 - fold enhancement of k + currents in wild - type larvae ( y . zhong et al ., neuron 14 : 527 ( 1995 ); y . zhong , nature 375 : 588 ( 1995 )). in nf1 p1 and nf1 p2 mutants , the inward current remained mostly intact , but the enhancement of k + currents was abolished . because the inward current is not affected in nf1 mutants , it appears that pacap38 receptors are normally activated by the peptide in these mutants . to rule out potential developmental effects of the nf1 mutation , transgenic flies carrying an inducible normal nf1 gene were studied . the hsnf1 transgene was expressed after heat shock in transgenic nf1 mutants , hsnf1 ; nf1 p1 and hsnf1 ; nf1 p2 . pacap38 - induced enhancement of k + currents was observed in hsnf1 ; nf1 p1 larvae subjected to heat shock ( 37 ° c . for 1 hour ) and not in those without heat shock . hsnf1 ; nf1 p2 larvae , however , showed a normal response to pacap38 even in larvae not subjected to heat shock . this was probably the result of constitutive expression of the hsnf1 transgene because a large amount of nf1 protein was detected in these flies . to reduce the amount of hsnf1 expression , hsnf1 ; nf1 p2 /+; nf1 p2 larvae were selected in which only one copy of the hsnf1 transgene was present . in these larvae , the pacap38 response was only observed after heat shock . the pacap38 - induced enhancement was fully rescued 4 hours after heat shock , but was observed with a smaller enhancement as early as 1 . 5 hours after heat shock . such a time course suggests that all other components in the pacap38 signaling pathways remain intact so that the preparation resumes pacap38 responsiveness as soon as enough nf1 protein is synthesized . because pacap38 is a vertebrate peptide ( a . arimura , regulatory peptides 37 : 287 ( 1992 ); d . spengler et al ., nature 365 : 170 ( 1993 )), the response induced by endogenous pacap38 - like neuropeptide was tested ( y . zhong et al ., neuron 14 : 527 ( 1995 ); y . zhong , nature 375 : 588 ( 1995 )). high frequency stimulation ( 40 hz ) applied to motor axons through a suction pipette increased k + currents , presumably by causing release of pacap38 - like peptides ( y . zhong et al ., neuron 14 : 527 ( 1995 )). this evoked pacap3 8 - like response was also eliminated in nf1 mutants and rescued by the expression of the hsnf1 transgene . because the nf1 protein acts as a ras - gap ( g . f . xu , et al ., cell 62 : 599 ( 1990 ); cell 63 : 835 ( 1990 ); a . m . buchberg et al ., nature 347 : 291 ( 1990 ); r . ballester et al ., cell 63 : 851 ( 1990 ); g . a . martin et al ., cell 63 : 843 ( 1990 )), two null alleles of drosophila gap1 , ri 533b1 and ri 533pb were examined . flies carrying the mutations have disrupted eye development that results from increased ras activity ( u . gaul et al ., cell 68 : 1007 ( 1992 )). pacap38 induced a normal enhancement of k + currents in both gap1 mutants . moreover , recordings from transgenic larvae showed that induced expression of constitutively active ras ( ras v12 ) ( m . e . fortini et al ., nature 355 : 559 ( 1992 )) or active raf protein kinase ( raf gof ) ( a . h . brand et al ., genes & amp ; development 8 : 629 ( 1994 )) neither blocked nor mimicked the pacap38 response ( y . zhong , nature 375 : 588 ( 1995 )). these results suggest that failure to negatively regulate ras - raf signaling does not explain the defective pacap38 response in nf1 mutants . application of the membrane permeable camp analogs , dibutyryl camp or 8 - bromo camp to the larval neuromuscular preparation is insufficient to produce the pacap38 - like enhancement of k + currents ( y . zhong , nature 375 : 588 ( 1995 ); y . zhong et al ., j . neurogenet . 9 : 15 ( 1993 )) and appeared not to disrupt the pacap38 response in wild - type larvae . this implies that camp may not cause inhibition of the raf activity as reported in other preparations ( s . j . cook et al ., science 262 : 1069 ( 1993 )). application of these camp analogs to nf1 mutants did restore the normal response to pacap38 . both nf1 p2 homozygotes and heteroallelic nf1 p1 / nf1 p2 larvae showed enhanced k + currents . nf1 p1 larvae also responded , but with a smaller amplitude of response , which may be a non - specific effect of genetic background because the response of nf1 p1 / nf1 p2 heterozygotes to pacap38 was fully restored by treatment with camp analogs . the camp analogs were effective if applied any time before or within 2 min . after applying pacap38 . after 2 min , camp analogs failed to enhance the response of nf1 p2 mutants to pacap38 . this time course is consistent with a model whereby in nf1 mutants , the ras - raf pathway is normally activated in response to pacap38 for 2 min , but the camp pathway is blocked . therefore , synergistic modulation of k + currents can be achieved if camp analogs are supplied during the transient activation of the ras - raf pathway . addition of camp analogs also restored the response to pacap38 in rut 1 mutants , but not in ras 12a mutants ( m . s . livingstone et al ., cell 37 : 205 ( 1984 ); l . r . levin et al ., cell 68 : 479 ( 1992 ); y . zhong , nature 375 : 588 ( 1995 )). to further test whether activation of camp signaling rescues the defective pacap response of nf1 mutants , the drug forskolin , which stimulates g - protein coupled adenylyl cyclase activity ( k . b . seamon et al ., j cyclic nucleotide res . 22 : 201 ( 1981 ); y . dudai et al ., j neurogenet . 2 : 365 ( 1985 )), was applied to the neuromuscular preparation . pacap38 induced a normal response in nf1 p2 and nf1 p1 / nf1 p2 mutants exposed to forskolin . this indicates that adenylyl cyclase is present , but is not activated by receptors for pacap38 - like neuropeptides . forskolin also restored the pacap38 response in rut 1 mutants even though the rut - adenylyl cyclase is completely nonfunctional ( m . s . livingstone et al ., cell 37 : 205 ( 1984 ); l . r . levin et al ., cell 68 : 479 ( 1992 )). it is possible that camp synthesized by other adenylyl cyclases upon forskolin stimulation is sufficient to modulate k + currents together with the ras pathway activated by pacap38 ( m . s . livingstone , proc natl acad sci usa 82 : 5992 ( 1985 )). adenylyl cyclase shows abnormal subcellular localization in yeast ira mutants ( m . r . mitts et al ., mol cell biol 11 : 591 , ( 1991 )). the ira gene encodes proteins that are distantly related to the nf1 protein and that are involved in mediating ras - dependent activation of adenylyl cyclase . although the yeast cyclase is very different from rut - adenylyl cyclase and other cyclases in higher organisms ( t . toda et al ., cell 40 : 27 ( 1985 ); k . tanaka et al , cell 60 : 803 ( 1990 )), adenylyl cyclase activity in membrane fractions was examined . adenylyl cyclase activity was assayed as described ( m . s . livingstone et al ., cell 37 : 205 ( 1984 )) with membranes from abdomens of rut 1 , nf1 p2 , and wild type flies . calcium concentrations were calculated according to maxchelator v1 . 31 ( d . m . bers et al ., methods cell . biol . 40 : 3 ( 1994 )). assays were done in duplicate and each result represents data from at least two separate experiments . the rut - adenylyl cyclase is the only cyclase that can be activated by ca 2 + - calmodulin ( cam ) in the tissues from fly abdomen , as indicated by the lack of the ca 2 + - dependent cyclase activity in rut 1 mutants ( m . s . livingstone et al ., cell 37 : 205 ( 1984 ); l . r . levin et al ., cell 68 : 479 ( 1992 )). in addition , the basal activity ( m . s . livingstone et al ., cell 37 : 205 ( 1984 )) and the forskolin - stimulated ( y . dudai et al ., j neurogenet . 2 : 365 ( 1985 )) activity of adenylyl cyclase were also reduced in rut 1 mutants . however , nf1 mutations did not affect the basal activity , the ca 2 + - dependent activity , or the forskolin - stimulated activity of adenylyl cyclase . therefore , rut - adenylyl cyclase is present in these membranes and can be normally activated by ca 2 + - cam and forskolin . while this invention has been particularly shown and described with references to preferred embodiments thereof , it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims . those skilled in the art will recognize or be able to ascertain using no more than routine experimentation , many equivalents to the specific embodiments of the invention described specifically herein . such equivalents are intended to be encompassed in the scope of the claims . met thr gln lys pro gly glu trp ala ser ala leu leu ala arg phe glu asp gln leu pro asn arg ile gly ala tyr gly thr gln ala arg met ser gln asp gln leu val ala cys leu ile his ile ser arg tyr arg phe ser leu val ile ser gly leu thr lys met leu gln arg val asn glu ala ala leu gln asn arg his glu pro glu arg cys tyr phe gln thr lys asp thr ala arg phe glu glu ala met asn val lys leu ile gln glu leu thr ser cys ser glu glu asn pro asp tyr asn asp leu leu gln glu thr ile thr lys phe arg ser lys arg ala pro pro tyr his pro gln glu phe gln asp leu gln arg gly thr asn arg asp ile ser thr cys trp glu pro leu met asp phe val glu tyr phe lys thr glu asn lys lys ser lys thr leu val trp pro leu gln met leu phe ile glu ser ile lys arg gly leu gly gln his ser pro ser lys lys pro phe ser arg gly gln gly tyr asn phe ala asp ile glu leu met ile asp cys trp val ser cys phe arg ile asn pro his asn ile glu 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ser asn glu lys ile ser thr gln leu tyr pro ile leu phe asp gln val arg ala ile val ile asn thr gln phe ile glu his thr ile tyr ile met lys ser ile asp leu ala phe arg gln glu met lys phe arg asn lys leu val glu tyr leu thr asp trp val met gly thr ser his gln ile ala pro pro phe glu gln leu val gln leu val thr met ile ser asp lys gly glu leu ser pro leu leu trp asn met phe tyr arg glu val glu val ser asp cys met gln thr leu phe arg gly asn ser leu gly ser lys ile met ala phe cys phe lys ile tyr gly ala ser tyr leu gln met leu phe glu val asp pro ala arg leu asp pro thr glu asp ile glu gln his arg asn asn leu ile ala leu thr gln lys val phe asp ala ile ile asn ser ser asp arg phe pro pro gln leu arg ser met cys his cys leu tyr gln val leu ser lys arg phe pro asn leu leu gln asn ala ile val ser pro gln glu leu gly ile val asp lys gln val his ile ala asn his val glu phe ser lys glu gln his met leu cys phe gln ile ala ser asp cys glu thr val asp gln thr ser his ser met ser phe ile ser asp ala asn val leu ala leu his arg leu leu trp thr his gln glu lys ile gly asp tyr leu ser ser ser arg asp his lys ala val gly arg arg pro phe asp lys met ala thr leu leu ala tyr leu gly pro pro glu his lys pro val asp ser his met met phe ser ser tyr ala arg trp ser ser ile asp met ser ser thr asn phe thr leu lys ser met asn ile phe tyr gln ala gly thr ser lys ser thr asn gly asp leu leu ile tyr his val ile leu thr leu lys pro ser asp asn arg phe arg thr glu phe leu gln lys trp phe tyr val cys asn ser trp val arg glu tyr thr lys phe his asp arg ile leu lys leu thr asp phe ile asp ala glu gln gln lys leu pro gly ala leu ser his lys asp thr lys val ala ile lys val gly pro thr ala val asp asp asn gln phe thr leu ser ile thr asn glu ser gly gln leu ser phe ile his asn asp cys asp asn ile val gln ala ile ile his ile arg asn arg trp glu leu ser gln pro asp ser val thr val his gln lys ile arg pro lys asp val pro gly thr leu leu asn met tyr asn leu leu cys ala leu thr ala thr phe asp leu lys ile glu ile phe ile lys ser val ser glu lys leu ala thr asn glu pro his thr ile glu leu lys his leu cys leu glu tyr met thr pro trp leu lys glu met tyr pro ser val gln ala lys ile trp gly ser ile gly gln ile pro glu leu ile asp met val leu asp asn phe leu his lys ser ile thr tyr gly leu gly ser pro gln val glu ile met ala asp val ile thr arg ile cys arg val met asp lys ser cys thr asn pro thr gln tyr leu glu gln his met met trp asp asp ile ala ile leu gly arg tyr leu leu met leu ser phe asn asn cys leu asp val ala gly ser leu ser met arg ala ser thr his gly leu val ile asn ile ile his ser leu cys thr cys thr asn pro ser phe ser glu glu ala phe arg ser ser cys arg his pro thr asp lys trp leu gly asn glu met arg asp val pro asp cys glu trp leu asn thr trp thr ser leu leu ile val tyr gly cys ile ser lys ser val thr asp his glu val lys gln leu leu arg ile leu val lys ala leu glu ser phe asn asp leu ile leu ile glu ala leu val met cys leu thr arg ile gln pro leu leu arg pro glu ser pro ile his arg ala leu phe trp val ala ile ser val leu gln leu asp glu ile thr leu tyr gly ala gly leu ala leu leu glu gln asn leu his thr leu lys ser gln gly cys phe leu glu trp his phe lys gln leu asp his ala val gly leu ser phe arg ser asn phe his phe ala leu val gly his leu ile lys gly phe met leu leu gly ile tyr ala lys pro leu his arg asp lys phe glu glu glu val arg ser arg cys his val lys his ala leu pro arg trp val gln ala ile gly leu pro leu ser arg arg gln lys ser trp asp ile leu asp gln ser ala leu gln phe ala arg gln his lys val pro val pro thr thr lys asp pro asn asn ala thr gly ile glu glu arg arg val leu tyr gln tyr leu ala glu gly ser val val phe pro lys val phe pro val ile his ser leu leu asp gln lys ile asn asn ile ile gln asn met leu ala ser glu asp pro ser gln gln gln leu his phe thr lys tyr asn met met gly glu ser ser glu leu phe val asn cys leu glu ala met val glu thr cys leu pro gly asp glu ser ala leu asp phe tyr asp asn cys pro gly ser val ser ser leu arg arg phe asp glu gln leu pro ile lys thr gly gln gln asn thr his thr val asn asn met arg ile phe gly glu ala ala glu lys asn leu tyr leu ser gln leu ile ile leu asp thr leu glu lys cys leu ala gly leu leu pro glu ile cys his phe leu his thr cys arg glu gly asn gln his ala ala glu leu arg asn ser ala ser gly val leu phe ser ile glu leu leu gln tyr ile asn val asp cys ala lys leu lys arg gln leu ala val ile asn ser leu glu lys ala phe trp asn trp val glu asn tyr pro asp glu phe thr lys leu tyr gln ile pro gln thr ala glu ser thr lys arg lys ala ala val trp pro leu gln ile ile ala ile ala cys val lys leu cys lys ala ser thr tyr ile asn trp gln pro ala asp val asp leu met ile asp cys leu val ser cys phe arg ile ser pro his asn asn gln his phe lys ile cys leu ala gln ile ile thr asn ser ala leu asp trp trp pro lys ile asp ala val tyr cys his ser val glu leu arg asn met phe gly glu thr leu his lys ala val gln gly cys gly ala his pro ala ile arg met ala pro val lys leu ile his ala asp pro lys leu leu leu cys asn pro arg trp asn pro asp ala pro val glu thr phe trp glu ile ser ser gln met leu phe tyr ile cys lys lys leu thr ser his gln met leu ser lys phe leu leu lys asn lys gln ala asp arg ser ser cys his phe leu leu phe tyr gly val gly cys asp ile pro ser ser gly asn thr ser gln met ser met asp his glu glu leu leu arg thr pro gly ala cys ser gly thr pro pro ile cys arg gln ala gln thr lys leu glu val ala leu tyr met phe leu trp asn pro asp thr glu ala val leu val ala met ser cys phe arg his leu cys glu glu ala asp ile arg cys gly val asp glu val ser val his asn leu leu pro asn tyr asn glu gln ala thr lys leu ile leu asn tyr pro lys ala lys met glu asp gly gln ala ala glu ser leu his lys thr ile val lys arg arg ser leu gln glu trp ile asn met thr gly phe leu cys ala leu gly gly val cys leu gln gln arg ser asn ser gly leu ala thr tyr ser met ser ser glu gly asn ala asp thr pro val ser lys phe met asp arg leu leu ser leu met val cys asn his glu lys val gly leu gln ile arg thr asn val lys asp 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gln ser ile ala asn his val leu phe thr lys glu glu his met phe phe leu asp ile ala ser asp cys pro thr ser asp ala val asn his ser leu ser phe ile ser asp gly asn val leu ala leu his arg arg asp his lys ala val gly arg arg pro phe asp lys met ala thr leu leu ala tyr leu gly pro pro glu his lys pro val ala asp thr leu ser ile phe tyr gln ala gly thr ser lys ala gly asn pro ile phe tyr tyr val ala arg arg phe lys thr gly gln ile asn gly asp pro tyr glu ile val val asp leu thr his thr gly pro ser asn arg gly ser lys arg leu val phe ile asp cys pro gly lys leu ala glu ser ala glu arg thr lys val leu gly gln ser val phe leu asn asp arg trp glu leu ser gln pro asp ser ile pro gln his thr lys ile arg pro lys asp val pro gly thr leu leu asn ile ala leu leu asn cys ala leu thr cys thr phe asn leu lys ile glu gly gln leu leu glu thr ser gly leu cys ile pro ala asn asn thr leu phe ile val lys his leu cys leu glu tyr met thr pro trp leu ser asn leu val arg phe cys lys his asn asp asp ala lys arg gln arg val thr ala pro ser ile gln ala lys ile trp gly ser leu gly gln ile thr asp gly leu gly ser ile lys ala glu val met ala asp thr ala val ala glu gln his leu met trp asp asp ile ala ile leu ala arg tyr met glu ala cys met arg asp ile pro thr cys lys trp leu asp gln trp thr glu leu ala gln arg phe ala phe gln tyr asn pro ser leu gln pro arg ala leu val val phe gly cys ile ser lys arg val ser his cys leu lys gly pro asp thr tyr asn ser gln val leu ile glu ala thr val ile ala leu thr lys leu gln pro leu leu asn lys asp ser asp glu val asn leu tyr ser ala gly thr ala leu leu glu gln asn leu his thr leu asp ser leu arg ile phe asn asp lys ser pro glu glu val phe met ala ile arg asn pro leu glu trp his cys lys gln leu val gly his leu leu lys gly tyr arg his pro ser pro ala ile lys his arg asn cys asp lys phe glu val asn thr gln ser val ala asn val pro met asp thr tyr pro ile his his gly asp pro ser tyr ala arg lys ser met ser leu asp met gly gln pro ser gln ala asn asp thr lys ala pro lys arg gln glu met glu ser gly ile thr thr leu asn pro ile his gly ile val gln ser val val tyr his glu glu gly leu trp arg phe ala gly pro phe ser lys gln thr gln ile pro his ser pro gly ile asp lys glu asn val glu leu ser pro thr thr