West, Anthony Patrick; (1992) Regulation of the luteinizing hormone receptor in leydig cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The structure-function relationships and regulation of luteinizing hormone (LH)/chorionic gonadotropin (CG) receptors in Leydig cells was investigated. Four Leydig cell types were used: two mouse tumour Leydig cells (MA10 and MLTC-1), a rat tumour Leydig cell (R2C) and rat testis Leydig cells. Desensitization, up- and down-regulation of receptors were studied including the role of proteolysis in the mechanism of down-regulation. A new method was developed to study these processes, which generates truncated, but functional receptors using antisense oligodeoxynucleotides. The up-regulation of functional LH/CG receptors was found to occur only in MA10 cells, which continually synthesize their LH/CG receptor, after a 24h incubation. This is a cyclic AMP-dependent mechanism and occurs at low doses of agonists. The increase was inhibited by cycloheximide and actinomycin D. The mechanism for the loss of binding sites in Leydig cells over 2h was found to be different in the rat and mouse Leydig cells. No loss of receptors was observed in rat Leydig cells, but continual proteolysis of LH/CG receptors occurred at the plasma membrane. In mouse Leydig cells LH-induced loss of binding sites was due to a cyclic AMP-dependent mechanism - which occurred at high doses of agonists and resulted in an inhibition of internalization allowing for loss of binding sites at the plasma membrane by proteolytic cleavage. Using antisense coligodeoxynucleotides, truncated but functional receptors in the MA10 cell were generated. Six different antisense oligodeoxynucleotides were used to show that the phosphorylation sites on the intracellular C-terminal tail are required for desensitization. Furthermore the conformation of the 7th transmembrane domain and intracellular C-terminal tail were shown to be important for the mechanism of desensitization. Also, the final 9 amino acids on the intracellular C-terminal tail are essential for LH-induced loss of receptors. MA10 cells secrete high affinity LH/CG binding proteins, which were shown to be coded for by LH/CG receptor mRNA.

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