The interaction of nucleoside diphosphate kinase B with Gβγ dimers controls heterotrimeric G protein function

Abstract

Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein ␤␥ dimer (G␤␥) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in G␤ 1␥2 dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating G␣ s and G␣i subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish G␤ 1 orthologs, G␤1 and G␤1like, causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of G␤ 1/G␤1like is associated with a down-regulation in caveolins, AC-regulating G␣-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with G␤␥ dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.cAMP ͉ cardiac contractility ͉ G proteins ͉ NDPK ͉ zebrafish S ignaling through the activation of G proteins represents the most widely used signaling pathway in mammalian biology (1). A variety of G protein-coupled receptors (GPCRs) mediate extracellular signals via heterotrimeric G proteins, which are composed of a guanine nucleotide binding ␣-subunit (G␣), as well as a ␤-subunit (G␤) and a ␥-subunit (G␥). Upon GPCR activation, the bound GDP in G␣ is exchanged for GTP and both the GTP-liganded G␣ and the stable dimer G␤␥ regulate downstream effectors (2).Nucleoside diphosphate kinases (NDPKs), which catalyze the transfer of ␥-phosphate between NTPs and NDPs, represent a family of multifunctional proteins encoded by nine human nm23 genes. The two major isoforms, NDPK A and B (17-21 kDa), play crucial roles in a wide array of cellular processes [for review see (3)]. Despite their high sequence homology and the well known formation of heterohexamers to perform their house keeping enzyme activity (4), NDPK A and B have distinct cellular functions, which are based on the possibility of both isoforms contributing to multimeric protein complexes like the SET complex (5) and a complex formed with Ca 2ϩ -activated potassium channel KCa3.1 (6). In such complexes, NDPK not only supplies NTPs but also acts as protein kinase (6). We have previously shown that NDPK B, but not NDPK A, forms a complex with G␤␥ (7, 8) and acts as a histidine kinase for G␤. The high energetic phosphate on G␤ can be specifically transferred to GDP and the GTP that is formed, induces G protei...

Regulation of heterotrimeric G-protein signaling by NDPK/NME proteins and caveolins: an update

“…Knockdown of NDPK-C also reduces cAMP production, which is consistent with the essential role of NDPK-C mediating the interaction between G proteins and NDPK-B. 39 Knockdown of NDPK-B in zebrafish also reduces G-protein expression, with a similar phenotype as the Gβ 1 /Gβ 1-like knockdown zebrafish, 41 suggesting that, in addition to acute regulation of G-protein activity, long-term modulation of NDPKs may influence the protein levels of G proteins, possibly because of the membrane-targeting effects of NDPK complexes. Consistently, studies in neonatal rat cardiomyocytes have shown that NDPK-B and NDPK-C critically regulate the membrane localization of G proteins.…”

“…Consistently, studies in neonatal rat cardiomyocytes have shown that NDPK-B and NDPK-C critically regulate the membrane localization of G proteins. 39,41 Moreover, acute stimulation of rat cardiomyocytes overexpressing NDPK-C with the β-adrenoceptor agonist isoprenaline results in enhanced membrane localization of G proteins and NDPK-C, 39 suggesting that recruitment of NDPK/G protein complexes to the plasma membrane may counteract a fading response to long-term isoprenaline stimulation due to desensitization of β-adrenoceptors.…”

Nucleoside diphosphate kinases (NDPKs) in animal development

“…Re-expression of human NME2 in KO MEFs rescued the loss in G protein content and restored basal cAMP levels [104]. The Wieland laboratory showed that heart failure is associated with similar defects and in zebrafish, where morpholino-mediated knockdown of NME2, as well as Gb1, both resulted in dramatically reduced cardiac contractility (Fig.…”

“…1d). They suggested that decreased asubunit abundance alters adenylyl cyclase-regulating Gs and Gi protein subfamilies [104]. These defects were also rescued by injection of zebrafish Nme2 mRNA into morphants.…”

“…-permeable channel, the transient receptor potential-vanilloid-5 (TRPV5) channel was found to be phosphorylated and thus activated by NME2 [109]. Experiments performed on fibroblasts of Nme1 -/-/ Nme2 -/-knockout mice and zebrafish show that NME2 influences heterotrimeric G protein signaling through phosphorylation of Gbc dimers, thereby stimulating cardiac contractility [104]. Data on the activation of heterotrimeric G proteins and different ion channels point to a role of NDPK as a histidine phosphotransferase (histidine kinase).…”