Growth Hormone-induced Diacylglycerol and Ceramide Formation via Gαi3 and Gβγ in GH4 Pituitary Cells: POTENTIATION BY DOPAMINE-D2 RECEPTOR ACTIVATION

Gele Liu,Liliane Robillard,Behzad Banihashemi,Paul R Albert

doi:10.1074/jbc.m202130200

Abstract

Growth hormone (GH) secretion is regulated by indirect negative feedback mechanisms. To address whether GH has direct actions on pituitary cells, lipid signaling in GH(4)ZR(7) somatomammotroph cells was examined. GH (EC(50) = 5 nm) stimulated diacylglycerol (DAG) and ceramide formation in parallel by over 10-fold within 15 min and persisting for >3 h. GH-induced DAG/ceramide formation was blocked by pertussis toxin (PTX) implicating G(i)/G(o) proteins and was potentiated 1.5-fold by activation of G(i)/G(o)-coupled dopamine-D2S receptors, which had no effect alone. Following PTX pretreatment, only PTX-resistant Galpha(i)3, not Galpha(o) or Galpha(i)2, rescued GH-induced DAG/ceramide signaling. GH-induced DAG/ceramide formation was also blocked in cells expressing Gbetagamma blocker GRK-ct. In GH(4)ZR(7) cells, GH induced phosphorylation of JAK2 and STAT5, which was blocked by PTX and mimicked by ceramide analogue C2-ceramide or sphingomyelinase treatment to increase endogenous ceramide. We conclude that in GH(4) pituitary cells, GH induces formation of DAG/ceramide via a novel Galpha(i)3/Gbetagamma-dependent pathway. This novel pathway suggests a mechanism for autocrine feedback regulation by GH of pituitary function.

Highlights

Growth hormone (GH) secretion is regulated by indirect negative feedback mechanisms
The contribution of specific G␣ subunits to GH autocrine signaling pathways was addressed using PTXinsensitive mutants of G␣i2, G␣i3, and G␣o individually transfected into GH4ZR7 pituitary cells (GH4C1 cells transfected with the dopamine-D2S receptor [24, 25])
Concentration- and Time-dependent Increase in DAG/Ceramide Formation Induced by GH—The acute action of GH on endogenous DAG and ceramide levels in GH4ZR7 pituitary cells was assessed by the DAG kinase assay

Summary

Introduction

Growth hormone (GH) secretion is regulated by indirect negative feedback mechanisms. To address whether GH has direct actions on pituitary cells, lipid signaling in GH4ZR7 somatomammotroph cells was examined. GH-induced DAG/ceramide formation was blocked by pertussis toxin (PTX) implicating Gi/Go proteins and was potentiated 1.5-fold by activation of Gi/Go-coupled dopamine-D2S receptors, which had no effect alone. We conclude that in GH4 pituitary cells, GH induces formation of DAG/ceramide via a novel G␣i3/ G␤␥-dependent pathway. This novel pathway suggests a mechanism for autocrine feedback regulation by GH of pituitary function. The contribution of specific G␣ subunits to GH autocrine signaling pathways was addressed using PTXinsensitive mutants of G␣i2, G␣i3, and G␣o individually transfected into GH4ZR7 pituitary cells (GH4C1 cells transfected with the dopamine-D2S receptor [24, 25]). We have identified G␣i3 and G␤␥ as crucial for both GH-induced ceramide formation and dopamine-D2-induced potentiation of the GH response

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Conclusion