A total of 10 nucleotides of K1A5 appear to have been changed in “type”:”entrez-nucleotide”,”attrs”:”text”:”AY028961″,”term_id”:”13641197″,”term_text”:”AY028961″AY028961 (VL) by possible somatic mutations, resulting in 6 amino acid residue difference at aa 2, 3, 4, 7, 33, and 90. Thr-Leu-Pro-Gly or Ser-Leu-Pro-Gly (for the Drosophila serotonin transporter) was conserved in all of these transporter proteins. Open in a separate window Fig. 3 Structure alignment of the VH of K1-4c and of F11.2.32. The three-dimensional structure alignment was done using the MSA3D program in Advanced Protein Explorer. Within the variable region of the heavy chain (H), green indicates exact match between residues; light blue indicates similar match; yellow indicates difference; red indicates mismatch or insufficient sequence data. CDR 1, 2, 3: complementarity-determining region 1, 2, 3; P: peptide shown here in turquoise (PDB entry 2HRPP). Light chain can be seen in dark blue in the background. The VL of K1-4c belonged to the V IV family, subgroup II. The light chain CDR1 (aa 27 C 36), CDR2 (aa 55 C 57) and CDR3 (94 C 101) are located as shown in Fig. 4. Comparison of the VL with mouse germline V sequences showed that the nucleotide sequence was most homologous to K1A5 (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”D00081″,”term_id”:”220455″,”term_text”:”D00081″D00081) [22]. A total of 10 nucleotides of K1A5 appear to have been changed in “type”:”entrez-nucleotide”,”attrs”:”text”:”AY028961″,”term_id”:”13641197″,”term_text”:”AY028961″AY028961 (VL) by possible somatic mutations, resulting in 6 amino acid residue difference at aa 2, 3, 4, 7, 33, and 90. The amino acid sequence of the light chain CDR regions, except for one amino acid (aa 33: Thr instead of Asn) in CDR1, was identical to the sequence of the germline segment K1A5. The J gene segment of VL is identical to mouse germ line J5 gene segment (GeneBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”V00777″,”term_id”:”51657″,”term_text”:”V00777″V00777) [23]. Since anti-idiotypic response may depend on selection of non-germ line sequences [24], it is unlikely that the light chain of K1-4c significantly contributes to the specificity of this antibody. It is worth noting that many antibodies of discrete specificities, such Dicer1 as anti-GAT, anti-DNP, anti-flagellin, anti-phosphorylcholine, anti-digoxin, anti-phenyloxazolone, and anti-DNA, used almost the same V subgroup in association with various heavy chains [22]. Random pairing of a given V chain with distinct heavy chains would probably favor an escape from selection in evolution, especially Sulfacarbamide in view of the possible role of the idiotypic network acting as a built-in selection pressure [22]. The above observations suggest that the amino acid(s) (Ser/Thr-Leu-Pro-Gly) in the three or four positions close to TM5 of the DAT may contribute to the binding of cocaine. Further studies by point mutation or peptide mapping will be needed to confirm this prediction. We are currently generating the peptides from the antigen-combining region of K1-4c. We will examine whether indeed these peptides inhibit cocaine binding to the DAT without interfering with dopamine uptake. If this is so, these peptides may be used as novel cocaine antagonist peptides to study the interaction of Sulfacarbamide cocaine and the dopamine transporter as well Sulfacarbamide as potential therapeutic anti-cocaine addiction agents. Acknowledgments We thank Dr. Gary Olsen in Department of Microbiology for helpful advice in the analysis of sequence data, Lou Ann Miller in Center for Microscopic Imaging for assistance with microscopy photographs, and Dr. Elizabeth Greeley for critical reading of the manuscript. This work was supported in part by grant DA10367 from the National Institutes of Health. Abbreviations aaamino acid(s)bpbase pair(s)cDNADNA complementary to RNACDRcomplementarity-determining region(s)DATdopamine transporterhDAThuman dopamine transporterELISAenzyme-linked immunosorbent assayFRframework regionHheavy chainHIVhuman immunodeficiency virusIgimmunoglobulinLlight chainMWmolecular weightntnucleotide(s)PCRpolymerase chain reactionTMtransmembrane domainVvariable region.