Neuroscience Letters, 111 (1990) 351-356
351
Elsevier Scientific Publishers Ireland Ltd.
NSL 06775
Molecular cloning of human neuronal nicotinic receptor 3-subunit D. Fornasari, B. Chini, P. Tarroni and F. Clementi CNR Center of Cytopharmacology, Department of Medical Pharmacology, University of Milan, Milan (Italy) (Received 11 September 1989; Revised version received 14 November 1989; Accepted 11 December 1989)
Key words: Molecular cloning; Nicotinic receptor; Central nervous system; Neuroblastoma; Human Neuronal nicotinic receptors (nAchRs) have been isolated or cloned in insect, bird and mammalian neurons, but no information exists on the primary structure of human neuronal nAchRs. By screening a cDNA library from the human neuroblastoma cell line IMR 32 with a cDNA probe corresponding to the full length of rat ~t3-nicotinic subunit, we have identified an open reading frame encoding a protein of 502 amino acids. This protein shows all the features of members of the ligand-gated receptor superfamily and has two cysteine residues at positions 192, 193 which are typical of the nicotinic ~-subunits. Because of its high homology to rat ct3 (93% amino acid identity), we conclude that we have cloned the human ct3-nicotinic subunit.
Neuronal nicotinic receptors (nAchRs) are expressed in ganglia and in the central nervous system (CNS) [10] where they participate in different brain activities [6]. During the past few years, much information on the molecular and functional properties of neuronal nAchRs have become available [3, 8, 18]. These receptors are multimeric proteins constituted of two different subunits, ~ and fl, whose stoichiometry is still unclear. Several subtypes of ~- and fl-subunits have been isolated or cloned in insect, bird or mammalian neurons and the analysis of their structure has shown that they are members of the ligand-gated ion channel superfamily, which also includes glycine [5], GABAA [13] and muscle-type nAchR receptor subunits [15]. In spite of the possible involvement of neuronal nAchRs in human degenerative pathologies of the CNS, such as Alzheimer's and Parkinson's diseases [11], no data exist on the primary structure of the human neuronal nicotinic subunits and their localization in human brain. Indeed, the possibility to map single nAchR subunit subtypes in human brain is very attractive because it may allow the correlation of their expression with defined physio-pathological conditions. In order to get new insights into the molecular structure
Correspondence: D. Fornasari, CNR Center of Cytopharmacology, via Vanvitelli 32, 20129 Milano, Italy 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
352
and evolution of this receptor family and to generate more specific tools for in situ hybridization to human brain, we have studied several human neuroblastoma cell lines as possible sources of neuronal nicotinic receptors [14]. We focused our attention on IMR32, a cell line established in vitro by Tumilowicz [16]. In these cells we have previously shown, by electrophysiological recordings, the presence of nAchRs [4] and, by Northern analysis, m R N A s hybridizing to 2pCA48 (not shown), an approximately 2 kb e D N A probe containing the entire coding sequence of the rat ~3-subunit (1497 bp) [1]. (2pCA48 was a gift of Drs. J. Boulter and S. Heinemann.) This evidence induced us to screen a 2gtl 0 c D N A library prepared from the IMR32 cell line (kindly provided by Dr. H. Soreq), with the above-mentioned rat c D N A probe. Approximately 4 × 105 plaques were screened. Duplicate filters were hybridized to 106 cpm/ml of 32p radiolabelled probe in 50% formamide, 5 x SSC, at 42°C. Washings were carried out at a final stringency of 1 × SSC, at 70'~C. Ten strongly positive clones were identified, plaque purified and the phage D N A was isolated [9]. e D N A inserts were restricted out of the D N A , purified on low melt-agarose and recloned into the EcoRI site of MI3 mpl8. Single-stranded D N A in both orientations were obtained and sequenced by the chain termination method [12] using
...... C C C ACT C C C ~ A C C O T C C G
S e t Leu ~ u TCT C?G C ~
C~C
351
Elsevier Scientific Publishers Ireland Ltd.
NSL 06775
Molecular cloning of human neuronal nicotinic receptor 3-subunit D. Fornasari, B. Chini, P. Tarroni and F. Clementi CNR Center of Cytopharmacology, Department of Medical Pharmacology, University of Milan, Milan (Italy) (Received 11 September 1989; Revised version received 14 November 1989; Accepted 11 December 1989)
Key words: Molecular cloning; Nicotinic receptor; Central nervous system; Neuroblastoma; Human Neuronal nicotinic receptors (nAchRs) have been isolated or cloned in insect, bird and mammalian neurons, but no information exists on the primary structure of human neuronal nAchRs. By screening a cDNA library from the human neuroblastoma cell line IMR 32 with a cDNA probe corresponding to the full length of rat ~t3-nicotinic subunit, we have identified an open reading frame encoding a protein of 502 amino acids. This protein shows all the features of members of the ligand-gated receptor superfamily and has two cysteine residues at positions 192, 193 which are typical of the nicotinic ~-subunits. Because of its high homology to rat ct3 (93% amino acid identity), we conclude that we have cloned the human ct3-nicotinic subunit.
Neuronal nicotinic receptors (nAchRs) are expressed in ganglia and in the central nervous system (CNS) [10] where they participate in different brain activities [6]. During the past few years, much information on the molecular and functional properties of neuronal nAchRs have become available [3, 8, 18]. These receptors are multimeric proteins constituted of two different subunits, ~ and fl, whose stoichiometry is still unclear. Several subtypes of ~- and fl-subunits have been isolated or cloned in insect, bird or mammalian neurons and the analysis of their structure has shown that they are members of the ligand-gated ion channel superfamily, which also includes glycine [5], GABAA [13] and muscle-type nAchR receptor subunits [15]. In spite of the possible involvement of neuronal nAchRs in human degenerative pathologies of the CNS, such as Alzheimer's and Parkinson's diseases [11], no data exist on the primary structure of the human neuronal nicotinic subunits and their localization in human brain. Indeed, the possibility to map single nAchR subunit subtypes in human brain is very attractive because it may allow the correlation of their expression with defined physio-pathological conditions. In order to get new insights into the molecular structure
Correspondence: D. Fornasari, CNR Center of Cytopharmacology, via Vanvitelli 32, 20129 Milano, Italy 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
352
and evolution of this receptor family and to generate more specific tools for in situ hybridization to human brain, we have studied several human neuroblastoma cell lines as possible sources of neuronal nicotinic receptors [14]. We focused our attention on IMR32, a cell line established in vitro by Tumilowicz [16]. In these cells we have previously shown, by electrophysiological recordings, the presence of nAchRs [4] and, by Northern analysis, m R N A s hybridizing to 2pCA48 (not shown), an approximately 2 kb e D N A probe containing the entire coding sequence of the rat ~3-subunit (1497 bp) [1]. (2pCA48 was a gift of Drs. J. Boulter and S. Heinemann.) This evidence induced us to screen a 2gtl 0 c D N A library prepared from the IMR32 cell line (kindly provided by Dr. H. Soreq), with the above-mentioned rat c D N A probe. Approximately 4 × 105 plaques were screened. Duplicate filters were hybridized to 106 cpm/ml of 32p radiolabelled probe in 50% formamide, 5 x SSC, at 42°C. Washings were carried out at a final stringency of 1 × SSC, at 70'~C. Ten strongly positive clones were identified, plaque purified and the phage D N A was isolated [9]. e D N A inserts were restricted out of the D N A , purified on low melt-agarose and recloned into the EcoRI site of MI3 mpl8. Single-stranded D N A in both orientations were obtained and sequenced by the chain termination method [12] using
...... C C C ACT C C C ~ A C C O T C C G
S e t Leu ~ u TCT C?G C ~
C~C
