Insulin signaling inhibits the 5-HT2C receptor in choroid plexus via MAP kinase

Abstract

Background: G protein-coupled receptors (GPCRs) interact with heterotrimeric GTP-binding proteins (G proteins) to modulate acute changes in intracellular messenger levels and ion channel activity. In contrast, long-term changes in cellular growth, proliferation and differentiation are often mediated by tyrosine kinase receptors and certain GPCRs by activation of mitogen-activated protein (MAP) kinases. Complex interactions occur between these signaling pathways, but the specific mechanisms of such regulatory events are not well-understood. In particular it is not clear whether GPCRs are modulated by tyrosine kinase receptor-MAP kinase pathways.

Results: Here we describe tyrosine kinase receptor regulation of a GPCR via MAP kinase. Insulin reduced the activity of the 5-HT2C receptor in choroid plexus cells which was blocked by the MAP kinase kinase (MEK) inhibitor, PD 098059. We demonstrate that the inhibitory effect of insulin and insulin-like growth factor type 1 (IGF-1) on the 5-HT2C receptor is dependent on tyrosine kinase, RAS and MAP kinase. The effect may be receptor-specific: insulin had no effect on another GPCR that shares the same G protein signaling pathway as the 5-HT2C receptor. This effect is also direct: activated MAP kinase mimicked the effect of insulin, and removing a putative MAP kinase site from the 5-HT2C receptor abolished the effect of insulin.

Conclusion: These results show that insulin signaling can inhibit 5-HT2C receptor activity and suggest that MAP kinase may play a direct role in regulating the function of a specific GPCR.

Figure 1

Effect of insulin and PD 098059 on the response to serotonin in rat choroid plexus cells. (A) Change in intracellular calcium in response to 5-HT in a representative choroid plexus cell. Horizontal bar above the trace indicates bath application of 2 μM 5-HT. (B) Change in intracellular calcium in response to 5-HT after 10 minute treatment with insulin in a representative choroid plexus cell. Horizontal bars above the trace indicate bath application of 10 μM insulin or 2 μM 5-HT. (C) Change in peak intracellular calcium concentration in response to 5-HT with and without insulin treatment and/or PD 098059 pretreatment. Basal intracellular calcium levels averaged 20-50 nM for all groups. The data are presented as mean ± S.E.M. * indicates a statistical difference (P < 0.05) of the insulin treatment group when compared to control.

Figure 2

Effect of insulin and IGF-1 on the peak Cl^- current in response to serotonin or acetylcholine in Xenopus oocytes expressing 5-HT_2C and M1 acetylcholine receptors. (A) Effects of insulin or IGF-1 on the peak Cl^- current in response to 5-HT. Top left, representative Cl^- current trace after stimulation with 5-HT. Top right, representative Cl^- current trace after stimulation with 5-HT in an oocyte pre-treated with insulin. Horizontal bars above the current traces indicate bath application of 1 μM 5-HT. Middle, effects of insulin on the peak Cl^- current induced by 5-HT. Bottom, effects of IGF-1 on the peak Cl^- current induced by 5-HT. (B) Effects of insulin or IGF-1 on the peak Cl^- current in response to Ach. Top left, representative current trace after stimulation with Ach. Top right, representative current trace after stimulation with Ach in an oocyte previously treated with insulin. Horizontal bars above the current traces indicate bath application of 10 μM Ach. Middle, effects of insulin on the peak Cl^- current induced by Ach. Bottom, effects of IGF-1 on the peak Cl^- current induced by Ach. The data are presented as mean ± S.E.M. of 12-16 oocytes (*, P < 0.05).

Figure 3

Effects of tyrosine kinase inhibitor and dominant-negative RAS antibody on IGF-1 effects on the serotonin response in Xenopus oocytes. Shown are peak Cl^- currents in response to 5-HT after the indicated treatments. (A) Genistein, a tyrosine kinase inhibitor, blocks the inhibitory effect of IGF-1 on the 5-HT response. The data are presented as mean ± S.E.M. of 11-12 oocytes (*, P < 0.05). (B) RAS antibody, Y13-259, blocks the inhibitory effect of IGF-1 on the 5-HT response. The data are presented as mean ± S.E.M. of 6 oocytes (*, P < 0.05).

Figure 4

Role of MAP kinase in the inhibitory effect of IGF-1 on the 5-HT response in Xenopus oocytes. Plotted are peak Cl- currents in response to 5-HT after the indicated treatments. (A) The MEK kinase inhibitor, PD 098059, but not the protein kinase C inhibitor (PKC I), blocks the inhibitory effect of IGF-1 on the 5-HT response. The data are presented as the mean ± S.E.M. of 6 oocytes (*, P < 0.05). (B) Activated MAP kinase mimics the IGF-1 inhibitory effect on the 5-HT response. The data are presented as the mean ± S.E.M. of 8-12 oocytes (*, P < 0.05). (C) Mutation of the putative MAP kinase site on the 5-HT2C receptor abolishes the insulin effect. In oocytes expressing the wild-type 5-HT2C receptor (left two bars), insulin treatment resulted in a significant reduction of the peak Cl- current in response to 5-HT. In cells expressing the MAP kinase site mutation S159A (right two bars), insulin had no effect on the 5-HT-activated Cl- current. The data are presented as the mean ± S.E.M. of 6 oocytes (*, P < 0.05).