Nm23-H2 Interacts with a G Protein-coupled Receptor to Regulate Its Endocytosis through an Rac1-dependent Mechanism

Abstract

G protein-coupled receptors (GPCRs) represent a vast family of transmembrane proteins involved in the regulation of several physiological responses. The thromboxane A2 receptor (present as two isoforms: TP␣ and TP␤) is a GPCR displaying diverse pharmacological effects. As seen for many other GPCRs, TP␤ is regulated by agonistinduced internalization. In the present study, we report the identification by yeast two-hybrid screening of Nm23-H2, a nucleoside diphosphate kinase, as a new interacting molecular partner with the C-terminal tail of TP␤. This interaction was confirmed in a cellular context when Nm23-H2 was co-immunoprecipitated with TP␤ in HEK293 cells, a process dependent on agonist stimulation of the receptor. We observed that agonist-induced internalization of TP␤ was regulated by Nm23-H2 through modulation of Rac1 signaling. Immunofluorescence microscopy in HEK293 cells revealed that Nm23-H2 had a cytoplasmic and nuclear localization but was induced to translocate to the plasma membrane upon stimulation of TP␤ to show extensive co-localization with the receptor. Our findings represent the first demonstration of an interaction of an Nm23 protein with a membrane receptor and constitute a novel molecular regulatory mechanism of GPCR endocytosis.

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability

“…In contrast, an increase in NDPK content was not observed when hypertrophy was induced with thyroid hormone. Consistent with recent reports showing NDPK translocation by stimulation of GPCRs (Gallagher et al 2003;Rochdi et al 2004), these data suggest that sustained stimulation of β-adrenergic receptors increases association of NDPK with the plasma membrane. Taking the recent findings in the zebrafish into account, the primary regulation might occur at the level of mRNA encoding the interacting G protein α subunit (Müller et al 1994).…”

Molecular and Functional Interactions between Escherichia coli Nucleoside-diphosphate Kinase and the Uracil-DNA Glycosylase Ung

“…A large body of research also indicates that NM23/NDKs can interact directly with other proteins to regulate or provide links between different cellular pathways (17,18). Of note is the presence of NM23-H1/NDK-A/DNase in a DNA repair complex, where it interacts with the exonuclease TREX1 to degrade DNA during granzyme A-mediated cell death (11,19) as well as the interactions of NM23-H2/NDK-B with integrin (20) and with a membrane receptor protein (21). Whether the developmental and cancer-related functions of NM23/NDP kinases are driven by the chemical reactions they catalyze and/or by their protein/protein regulatory interactions remains to be elucidated.…”

Association of Nucleoside Diphosphate Kinase with Microtubule-Based Structures

“…In addition, NDPK B has also been shown to allow Gα q -induced internalization of the thromboxane A2 receptor (TPβ; Rochdi et al, 2004) by inactivating Rac1. The latter report potentially demonstrates an interaction between NDPK B and a GPCR signaling pathway.…”

“…In some cases, the recruitment of cytosolic NDPKs to membranes was ascribed to interactions with peripheral or integral membrane proteins such as the G protein transducin (Orlov and Kimura, 1998), integrin cytoplasmic domain associated protein 1-α (ICAP-1α; and the potassium channel K Ca 3.1 (Srivastava et al, 2006). Overexpression of cytosolic NDPKs revealed novel associations with the GTP-bound form of ARF6 , the thromboxane A2 β receptor (Rochdi et al, 2004) and vonHippel Lindau protein (Hsu et al, 2006), and these binding partners were proposed to anchor NDPK to membranes. However, in many instances the mechanism underlying the association of NDPKs with membranes remains unclear (e.g., Gallagher et al, 2003;Mizrahi et al, 2005).…”

“…Nevertheless, in many instances these proteins associate with membranes of a wide variety of intracellular compartments. Thus, cytosolic NDPKs are found at the plasma membrane where they are involved in the function of ion channels (Srivastava et al, 2006), receptor activation and desensitization by agonists Rochdi et al, 2004), activation of phagocyte NADPH oxidase (Mizrahi et al, 2005), dynamin-mediated endocytosis Palacios et al, 2002;Hsu et al, 2006) and integrinmediated adhesion . Soluble NDPKs also localize to endosomes (Hsu et al, 2006), membranes from the endoplasmic reticulum Kapetanovitch et al, 2005), the Golgi and vesicles budding from the trans-Golgi network , as well as phagosomes .…”