Role of disulphide and sulphydryl groups in clustering of enkephalin receptors in neuroblastoma cells

Abstract

Image intensified fluorescence microscopy has been very useful in visualising the patterns and mobility of receptors for epidermal growth factor (EGF), insulin and α₂-macroglobulin in intact cells^1,2. Recently, we have synthesised a bioactive derivative of enkephalin³, Tyr-D-Ala-Gly-Phe-Leu-Lys-rhodamine, and used it for the microscopic visualisation and localisation of opiate (enkephalin) receptors in neuroblastoma cells⁴. In contrast to the case of polypeptide hormones^1,2, where fluorescently labelled receptors are initially distributed uniformly and quickly form patches which are subsequently internalised^1,2, the enkephalin-labelled receptor patches in neuroblastoma cells appear only slowly and are not internalised⁴. Sulphydryl groups are involved in the binding of opiates to brain membranes and may affect differentially the binding of agonists and antagonists^5,6. Also, sulphydryl and disulphide groups are involved in the binding sites of adrenergic, cholinergic and muscarinic receptors^7–11. These observations prompted us to examine the effects of disulphide and sulphydryl reagents on the clustering of opiate (enkephalin) receptors in N4TG1 neuroblastoma cells. We report here that in these cells there are reactive sulphydryl and disulphide groups which are essential for cluster formation (but not binding), and that a sulphydryl–disulphide exchange reaction may be involved in this process. In addition, the sulphydryl reagents seem to dissociate the two steps of binding and cluster formation, and thus provide a tool for studying the pharmacological importance of receptor clustering.