Expression pattern of the type 1 sigma receptor in the brain and identity of critical anionic amino acid residues in the ligand-binding domain of thereceptor

The type I sigma receptor (sigma R1) has been shown to participate in a var iety of functions in the central nervous system. To identify the specific r egions of the brain that are involved in sigma R1 function, we analyzed the expression pattern of the receptor mRNA in the mouse brain by in situ hybr idization. sigma R1 mRNA was detectable primarily in the cerebral cortex, h ippocampus, and Purkinje cells of cerebellum. To identify the critical anio nic amino acid residues in the ligand-binding domain of sigma R1, we employ ed two different approaches: chemical modification of anionic amino acid re sidues and site-directed mutagenesis. Chemical modification of anionic amin o acids in sigma R1 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide redu ced the ligand-binding activity markedly. Since it is known that a splice v ariant of this receptor which lacks exon 3 does not have the ability to bin d a ligands, the ligand-binding domain with its critical anionic amino acid residues is likely to be present in or around the region coded by exon 3. Therefore, each of the anionic atnino acids in this region was mutated indi vidually and the influence of each mutation on ligand binding was assessed. These studies have identified two anionic amino acids, D126 and E172, that are obligatory for ligand binding. Even though the ligand-binding function was abolished by these two mutations, the expression of these mutants was normal at the protein level. These results show that sigma R1 is expressed at high levels in specific areas of the brain that are involved in memory, emotion and motor functions. The results also provide important information on the chemical nature of the ligand-binding site of sigma R1 that may be of use in the design of sigma R1-specific ligands with potential for modula tion of sigma R1-related brain functions. (C) 2001 Elsevier Science B.V. Al l rights reserved.