Belley M, Sullivan R, Reeves A, Evans J, O'Neill G, Ng G Y
Department of Chemistry, Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada.
Bioorg Med Chem. 1999 Dec;7(12):2697-704. doi: 10.1016/s0968-0896(99)00214-x.
A radioiodinated probe, [125I]-CGP 71872, containing an azido group that can be photoactivated, was synthesized and used to characterize GABA(B) receptors. Photoaffinity labeling experiments using crude membranes prepared from rat brain revealed two predominant ligand binding species at approximately 130 and approximately 100 kDa believed to represent the long (GABA(B)R1a) and short (GABA(B)R1b) forms of the receptor. Indeed, these ligand binding proteins were immunoprecipitated using a GABA(B) receptor-specific antibody confirming the receptor specificity of the photoaffinity probe. Most convincingly, [125I]-CGP 71872 binding was competitively inhibited in a dose-dependent manner by cold CGP 71872, GABA, saclofen, (-)-baclofen, (+)-baclofen and (L)-glutamic acid with a rank order and stereospecificity characteristic of the GABA(B) receptor. Photoaffinity labeling experiments revealed that the recombinant GABA(B)R2 receptor does not bind [125I]-CGP 71872, providing surprising and direct evidence that CGP 71872 is a GABA(B)R1 selective antagonist. Photoaffinity labeling experiments using rat tissues showed that both GABA(B)R1a and GABA(B)R1b are co-expressed in the brain, spinal cord, stomach and testis, but only the short GABA(B)R1b receptor form was detected in kidney and liver whereas the long GABA(B)R1a form was selectively expressed in the adrenal gland, pituitary, spleen and prostate. We report herein the synthesis and biochemical characterization of the nanomolar affinity [125I]-CGP 71872 and CGP 71872 GABA(B)R1 ligands, and differential tissue expression of the long GABA(B)R1a and short GABA(B)R1b receptor forms in rat and dog.
合成了一种含有可光活化叠氮基团的放射性碘化探针[¹²⁵I]-CGP 71872，并用于表征GABA(B)受体。使用从大鼠脑制备的粗膜进行的光亲和标记实验显示，在约130 kDa和约100 kDa处有两种主要的配体结合物种，据信分别代表受体的长形式（GABA(B)R1a）和短形式（GABA(B)R1b）。实际上，使用GABA(B)受体特异性抗体对这些配体结合蛋白进行了免疫沉淀，证实了光亲和探针的受体特异性。最有说服力的是，冷的CGP 71872、GABA、沙氯芬、(-)-巴氯芬、(+)-巴氯芬和(L)-谷氨酸以剂量依赖性方式竞争性抑制[¹²⁵I]-CGP 71872的结合，其排序和立体特异性具有GABA(B)受体的特征。光亲和标记实验表明，重组GABA(B)R2受体不结合[¹²⁵I]-CGP 71872，提供了令人惊讶的直接证据，证明CGP 71872是一种GABA(B)R1选择性拮抗剂。使用大鼠组织进行的光亲和标记实验表明，GABA(B)R1a和GABA(B)R1b在脑、脊髓、胃和睾丸中共同表达，但在肾脏和肝脏中仅检测到短的GABA(B)R1b受体形式，而长的GABA(B)R-1a形式在肾上腺、垂体、脾脏和前列腺中选择性表达。我们在此报告纳摩尔亲和力的[¹²⁵I]-CGP 71872和CGP 71872 GABA(B)R1配体的合成及生化表征，以及大鼠和犬中长的GABA(B)R1a和短的GABA(B)R1b受体形式的差异组织表达。
