J. Biol. Chem., Vol. 266, Issue 22, 14359-14366, 08, 1991

Regulation of the alpha 2A-adrenergic receptor in the HT29 cell line. Effects of insulin and growth factors

JC Devedjian, M Fargues, C Denis-Pouxviel, D Daviaud, H Prats and H Paris
Institut National de la Sante et de la Recherche Medicale U.317 Toulouse, France.

The density of the alpha 2A-adrenergic receptor in the HT29 cell line, a human colonic adenocarcinoma, increases when the cells are placed in fetal calf serum (FCS)-free culture medium and decreases again, in a concentration-dependent manner, when they are re-exposed to FCS. In an attempt to identify the FCS components responsible for this phenomenon, we examined the effect of insulin and of various growth factors on receptor expression. Incubation of HT29 cells with insulin resulted in a time- and dose-dependent lowering of the alpha 2-adrenergic receptor number. The decrease of [3H] RX821002 binding sites after a 48-h period of treatment reached 70-75% with 170 nM insulin, and a half-maximal effect was observed at 2.6 nM. This value is in agreement with the EC50 of the hormone for stimulating the glycolytic activity of HT29 cells (8 nM) and is sufficiently low to indicate that the decrease of alpha 2- adrenergic receptor number is mediated through stimulation of insulin receptors. Direct quantification of [3H] UK14304 binding sites and the study of the inhibition of [3H]RX821002 binding by (-)-epinephrine indicated that the degree of receptor coupling to Gi protein was not affected when the receptor number was decreased by insulin treatment. The reduction in receptor number did result in an attenuation of the inhibitory effect of UK14304 on forskolin-induced cAMP accumulation in a manner which was consistent with the existence of a large population of spare receptors in untreated cells. The action of insulin is not due to an accelerated rate of receptor degradation and can be mimicked by other growth factors (epidermal growth factor and insulin-like growth factors I and II) acting through stimulation of tyrosine kinase receptors. RNase mapping experiments with a 0.35-kilobase riboprobe prepared from the human alpha 2 C10-adrenergic receptor gene demonstrated that the decrease of receptor number induced by the different treatments is a reflection of changes occurring at the level of its mRNA. The use of cycloheximide indicated that the effect of insulin on alpha 2-adrenergic receptor mRNA does not require protein synthesis. The half-life of the alpha 2-adrenergic receptor mRNA measured after the addition of actinomycin D was unchanged by insulin which suggests that a decrease in the transcription rate is the predominant factor responsible for the observed regulation of receptor expression.
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