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The M581V Mutation, Associated with a Mild Form of Congenital Insensitivity to Pain with Anhidrosis, Causes Partial Inactivation of the NTRK1 Receptor

2002; Elsevier BV; Volume: 119; Issue: 4 Linguagem: Inglês

10.1046/j.1523-1747.2002.00140.x

1523-1747

Cláudia Miranda, Silvia Selleri, Marco A. Pierotti, Angela Greco,

Axon Guidance and Neuronal Signaling

To the Editor: Congenital insensitivity to pain with anhidrosis (CIPA; MIM 256800), also known as hereditary sensory and autonomic neuropathy type IV, is a rare autosomal recessive disorder associated with consanguinity (Axelrod and Pearson, 1984Axelrod F.B. Pearson J. Congenital sensory neuropathies. Diagnostic distinction from familial dysautonomia.Am J Dis Child. 1984; 138: 947-954Crossref PubMed Scopus (87) Google Scholar;Axelrod, 1996Axelrod F.B. Autonomic and sensory disorders.in: Emory A.E.H. Rimoin D.L. Principles of Medical Genetics. Churchill Livingstone, Edinburgh1996: 397-411Google Scholar) and is characterized by the absence of pain and temperature sensation and the lack of sweating. CIPA patients are generally children; they have recurrent episodes of unexplained fever, self-mutilation behavior, and occasionally mental retardation (Rosemberg et al., 1994Rosemberg S. Marie S.K. Kliemann S. Congenital insensitivity to pain with anhidrosis (hereditary sensory and autonomic neuropathy type IV).Pediatric Neurol. 1994; 11: 50-56Abstract Full Text PDF PubMed Scopus (158) Google Scholar). CIPA is the consequence of a genetic defect in the differentiation and migration of neural crest elements involved in nociceptive reception as well as in thermal regulation. Recently, mutations of the NTRK1 gene have been implicated in the pathogenesis of this disease. So far 37 mutations of NTRK1 have been detected in patients from different ethnic groups by several laboratories, including ours. Mutation types include frameshift, non-sense, splice site, and mis-sense (Indo, 2001Indo Y. Molecular basis of congenital insensitivity to pain with anhidrosis (CIPA): mutations and polymorphisms in TRKA (NTRK) (NTRK1) gene encoding the receptor tyrosine kinase for nerve growth factor.Hum Mutat. 2001; 18: 462-471Crossref PubMed Scopus (134) Google Scholar). The NTRK1 gene encodes one of the receptors of the nerve growth factor (NGF). NTRK1 protein is a tyrosine kinase receptor comprising an extracellular portion, involved in NGF binding, a single transmembrane region, a juxtamembrane domain, a tyrosine kinase domain, and a C-terminal tail. Most of CIPA mutations occur within the NTRK1 tyrosine kinase domain and a few within the extracellular domain. Recently, we analyzed the biologic effect of several NTRK1 germline mis-sense mutations and discovered at least three different pathogenic mechanisms responsible for the disease. Most of the mutations inactivate the NTRK1 receptor by completely abrogating its catalytic activity. The L213P mutation, in the extracellular domain, interferes with the receptor processing, causing its retention in the endoplasmic reticulum. The third mechanism, described for mutation D668Y, involves a reduction of the NTRK1 receptor activity, which is unlikely to be sufficient for the proper neuronal differentiation (Greco et al., 1999Greco A. Villa R. Tubino B. Romano L. Penso D. Pierotti M.A. A novel NTRK1 mutation associated with congenital insensitivity to pain with anhidrosis.Am J Hum Genet. 1999; 64: 1207-1210Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar,Greco et al., 2000Greco A. Villa R. Fusetti L. Orlandi R. Pierotti M.A. The Gly571Arg mutation, associated with the autonomic and sensory disorder CIPA, causes the inactivation of the NTRK1/NGF receptor.J Cell Physiol. 2000; 182: 127-133Crossref PubMed Scopus (30) Google Scholar;Miranda et al., 2002Miranda C. Di Virgilio M. Selleri S. Zanotti G. Pagliardini S. Pierotti M.A. Greco A. Novel pathogenic mechanisms of congenital insensitivity to pain with anhidrosis genetic disorder unveiled by functional analysis of neurotrophic tyrosine receptor kinase type1/nerve growth factor receptor mutations.J Biol Chem. 2002; 277: 6455-6462Crossref PubMed Scopus (36) Google Scholar). Recently the M581V mutation, occurring within subdomain V (β5 strand) of the NTRK1 tyrosine kinase domain, has been detected in a large Japanese CIPA family. The three affected individuals were adults and displayed milder clinical symptoms compared with other CIPA patients, including normal temperature sensation and a relatively long survival. Two patients were homozygous for the M581V mutation, whereas the third one was a compound heterozygous with a combination of M581V mutation and C1726 deletion (Yotsumoto et al., 1999Yotsumoto S. Setoyama M. Hozumi H. et al.A novel point mutation affecting the tyrosine kinase domain of the TRKA gene in a family with congenital insensitivity to pain with anhidrosis.J Invest Dermatol. 1999; 112: 810-814Crossref PubMed Scopus (22) Google Scholar). We investigated the effect of the M581V mutation on NTRK1 receptor activity. The mutation was introduced into the NTRK1 cDNA subcloned into the pRC/CMV mammalian expression vector, which carries the G418-resistance gene as a selectable marker. The NTRK1/M581V and NTRK1/wt constructs were transiently transfected into COS1 cells. Transfected cells were treated with or without NGF (50 ng per ml) for 10 min or 16 h, and cell extracts were immunoprecipitated with the MGR12 antibodies, which are specific for the receptor extracellular portion. Western blot analysis with anti-TRK antibodies (reacting with the intracellular NTRK1 portion) showed that the NTRK1/M581V receptor produced the 110 and 140 kDa forms indicating its proper expression (Figure 1a, upper panel). After western blot analysis with anti-phosphotyrosine antibodies, we detected a basal autophosphorylation of the wild-type receptor (due to the high expression level), which increased after NGF treatment. With respect to the NTRK1/M581V mutant, no phosphorylation was observed in untreated cells. NGF treatment induced a weak phosphorylation, highly reduced with respect to wild type, at 10 min and 16 h (Figure 1a, lower panel). The low tyrosine phosphorylation level achieved by NTRK1/M581V mutant after a long NGF treatment suggests a defect in the intrinsic kinase activity, rather than in the autophosphorylation kinetics. To investigate whether a deregulated phosphatase activity might account for the reduced phosphorylation of the NTRK1/M581V receptor, we treated transfected cells with sodium orthovanadate, a phosphatase inhibitor. The level of NTRK1/M581V receptor tyrosine phosphorylation increased in the presence of the inhibitor, and was detectable also in the absence of NGF stimulation (compare Figure 1b and a); however, the phosphorylation of the NTRK1/M581V receptor was significantly lower than wild type, both in the absence and in the presence of NGF, despite the similar protein expression levels. These data suggest that the M581V mutation interferes with the autophosphorylation of the NTRK1 receptor rather than with its downregulation by phosphatases. Most likely the M581V mutation introduces structural changes in the NTRK1 tyrosine kinase domain so that the receptor responsiveness is reduced. To determine the biologic effect of the M581V mutation we performed NIH3T3 transfection/focus forming assay, based on the ability of NGF-stimulated NTRK1 receptor to induce NIH3T3 transformation. This analysis has been successfully used to study the biologic activities of several NTRK1/CIPA mutants, and in particular to demonstrate the reduction of activity induced by the D668Y mutation (Greco et al., 1999Greco A. Villa R. Tubino B. Romano L. Penso D. Pierotti M.A. A novel NTRK1 mutation associated with congenital insensitivity to pain with anhidrosis.Am J Hum Genet. 1999; 64: 1207-1210Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar,Greco et al., 2000Greco A. Villa R. Fusetti L. Orlandi R. Pierotti M.A. The Gly571Arg mutation, associated with the autonomic and sensory disorder CIPA, causes the inactivation of the NTRK1/NGF receptor.J Cell Physiol. 2000; 182: 127-133Crossref PubMed Scopus (30) Google Scholar;Miranda et al., 2002Miranda C. Di Virgilio M. Selleri S. Zanotti G. Pagliardini S. Pierotti M.A. Greco A. Novel pathogenic mechanisms of congenital insensitivity to pain with anhidrosis genetic disorder unveiled by functional analysis of neurotrophic tyrosine receptor kinase type1/nerve growth factor receptor mutations.J Biol Chem. 2002; 277: 6455-6462Crossref PubMed Scopus (36) Google Scholar). Ten nanograms of NTRK1/wt, NTRK1/M581V, and NTRK1/D668Y plasmid DNA/2 × 105 cells were transfected; foci were selected in the absence or in the presence of 1 and 2 ng per ml of NGF. As shown in Figure 2, wild-type NTRK1 produced foci in the presence of both 1 and 2 ng per ml of NGF, in agreement with previous data (Miranda et al., 2002Miranda C. Di Virgilio M. Selleri S. Zanotti G. Pagliardini S. Pierotti M.A. Greco A. Novel pathogenic mechanisms of congenital insensitivity to pain with anhidrosis genetic disorder unveiled by functional analysis of neurotrophic tyrosine receptor kinase type1/nerve growth factor receptor mutations.J Biol Chem. 2002; 277: 6455-6462Crossref PubMed Scopus (36) Google Scholar). The M581V receptor produced foci only in the presence of 2 ng per ml of NGF, with strongly reduced efficiency with respect to the wild type. The transforming activity of D668Y mutant, used as a control, was significantly reduced with respect to the wild type, as previously reported (Miranda et al., 2002Miranda C. Di Virgilio M. Selleri S. Zanotti G. Pagliardini S. Pierotti M.A. Greco A. Novel pathogenic mechanisms of congenital insensitivity to pain with anhidrosis genetic disorder unveiled by functional analysis of neurotrophic tyrosine receptor kinase type1/nerve growth factor receptor mutations.J Biol Chem. 2002; 277: 6455-6462Crossref PubMed Scopus (36) Google Scholar), but slightly higher than M581V. We have shown that the M581V mutation causes a reduction of activity of the NTRK1 receptor and therefore we have identified an important residue for receptor activity. The molecular structure of NTRK1 receptor remains unsolved; however, a model of its inactive and active forms can be produced by analogy of the insulin receptor kinase. In this model methionine 581 is totally buried in a hydrophobic environment. Valine, despite being equally hydrophobic, is definitely smaller and could cause destabilization of this part of the molecule leading to a reduction in receptor responsiveness but not to complete inactivation. Thus, the residual activity may be sufficient for some of the NGF effects, and would explain the mild phenotype of the CIPA patients carrying the M581V mutation. In conclusion, our data indicate that a reduction in NTRK1 receptor activity may be a common mechanism in CIPA disease, being ascribed to two different mutations, the D668Y and the M581V, so far reported. This work was supported by Telethon Project nos E1159 and AIRC (Associazione Italiana Ricerca Cancro). The authors thank Dr Giuseppe Zanotti for helpful discussion, Miss Cristina Mazzadi for secretarial help and Dr Elda Tagliabue for kindly providing the MGR12 antibody.