Activation of Heterotrimeric G Proteins by a High Energy Phosphate Transfer via Nucleoside Diphosphate Kinase (NDPK) B and Gβ Subunits: SPECIFIC ACTIVATION OF Gsα BY AN NDPK B·Gβγ COMPLEX IN H10 CELLS

Hans-Joerg Hippe,Susanne Lutz,Friederike Cuello,Katrin Knorr,Achim Vogt,Karl H Jakobs,Thomas Wieland,Feraydoon Niroomand

doi:10.1074/jbc.m210305200

Abstract

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G(s)alpha and G(i)alpha proteins. However, co-expression of G(s)alpha led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to G(s)alpha. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gbetagamma dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gbeta subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B.Gbetagamma complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.

Highlights

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro
Co-expression of Gs␣ led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity
The amount of NDPK in cells expressing the inactive mutant H118N (M118) was increased 3-fold, whereas activity remained at the level found in untransfected cells

Summary

Introduction

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of Gs␣ and Gi␣ proteins. We have shown recently [10] that NDPK activates G proteins and regulates adenylyl cyclase activity in canine cardiac sarcolemmal membranes. This activation required the catalytic activity of NDPK (synthesis of GTP) but was clearly distinct from the effect of exogenous GTP, suggesting a more direct interaction of NDPK and G proteins

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