Interaction of the protein nucleobindin with G<sub>αi2</sub>, as revealed by the yeast two‐hybrid system

Mochizuki, Naoki; Hibi, Masahiko; Kanai, Yoshiyuki; Insel, Paul A.

doi:10.1016/0014-5793(95)01031-9

Cited by 45 publications

(29 citation statements)

References 26 publications

(17 reference statements)

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“…From these results obtained by two different approaches, we conclude that (i) calnuc specifically binds to G␣i3 in vivo, and (ii) the interaction is Ca 2ϩ ͞Mg 2ϩ -dependent but does not depend on the state of activation of G␣i3. The latter is in agreement with results previously reported by us (3) and others (9).…”

Section: Calnuc Specifically Interacts With G␣i3 and The Interaction Issupporting

confidence: 94%

“…Calnuc was previously demonstrated to interact with G␣ subunits, i.e., G␣i (3,9) and G␣s (3), and was found both in the cytosol and on Golgi membranes facing the Golgi lumen (3). Our previous studies focused on the membrane-associated pool of calnuc, which is the major Ca 2ϩ -binding protein in the Golgi and plays an important role in agonist releasable Ca 2ϩ storage in the Golgi (4).…”

Section: Discussionmentioning

confidence: 99%

“…Calnuc was found to interact with several different subfamilies of G␣ (3), including G␣i (3,9) and G␣s (3), using the yeast two-hybrid system. We further pinpointed the G␣i3 binding site on calnuc as the region containing both EF-hands (␣ helixloop-␣ helix) (3), which constitutes an ideal coiled-coil domain (10).…”

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confidence: 99%

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Calnuc, an EF-Hand Ca ²⁺ binding protein, specifically interacts with the C-terminal α5-helix of Gαi3

Lin

Fischer

Weiss

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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Calnuc (nucleobindin) was previously shown to be present both in the cytosol and in the Golgi and to be the major Golgi Ca 2؉ binding protein. In this study we verified the existence of the cytosolic pool of calnuc and investigated its interaction with G␣i3. Cytosolic calnuc was released by mild digitonin permeabilization. In pulsechase experiments, the two pools of calnuc had different mobilities, suggesting different posttranslational modifications. That calnuc interacts with G␣i3 in vivo was verified by the finding that G␣i3 could be crosslinked intracellularly to calnuc and co-immunoprecipitated from NIH 3T3 cells stably overexpressing either activated (Q204L) or inactivated (G203A) G␣i3. Binding was Ca 2؉ and Mg 2؉ -dependent. Calnuc and G␣i3-GFP codistributed primarily in the Golgi region. By yeast two-hybrid analysis, the binding site on G␣i3 for calnuc was mapped to the C-terminal region because removal of the last 12 amino acids (but not 11) abolished the interaction. Peptide competition indicated that calnuc, with its coiled-coil domain constituted by the two EF-hands, binds to G␣i3's C-terminal ␣5-helix. These results demonstrate that calnuc may play an important role in G protein-and Ca 2؉ -regulated signal transduction events.heterotrimeric G proteins ͉ nucleobindin ͉ signal sequence

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Section: Calnuc Specifically Interacts With G␣i3 and The Interaction Issupporting

confidence: 94%

Section: Discussionmentioning

confidence: 99%

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Calnuc, an EF-Hand Ca ²⁺ binding protein, specifically interacts with the C-terminal α5-helix of Gαi3

Lin

Fischer

Weiss

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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“…As is the case with all G protein-coupled receptors, mGluR2 activation causes GDP͞GTP exchange in its G protein targets and subsequent dissociation of the active GTP-bound G␣ and ␤͞␥ components of these heterotrimeric proteins. Activated G␣ i/o subunits inhibit particular isoforms of adenylate cyclase (7-9); however, additional actions are mediated through the actions of both G␣ and ␤͞␥ dimers on other targets (10)(11)(12)(13)(14)(15)(16). We generated transgenic mice that express a constitutively active form of G␣ i2 to test which of the effects of mGluR2 on mossy fiber synaptic plasticity can be accounted for by signaling mechanisms involving inhibitory G␣ subunits.…”

Section: Resultsmentioning

confidence: 99%

mGluR2 acts through inhibitory Gα subunits to regulate transmission and long-term plasticity at hippocampal mossy fiber-CA3 synapses

Nicholls¹,

Zhang²,

Bailey

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Presynaptic inhibitory G protein-coupled receptors play a critical role in regulating transmission at a number of synapses in the central and peripheral nervous system. We generated transgenic mice that express a constitutively active form of an inhibitory G␣ subunit to examine the molecular mechanisms underlying the actions of one such receptor, metabotropic glutamate receptor (mGluR) 2, at mossy fiber-CA3 synapses in the hippocampus. mGluR2 participates in at least three types of mossy fiber synaptic plasticity, (i) transient suppression of synaptic transmission, (ii) long-term depression (LTD), and (iii) inhibition of long-term potentiation (LTP), and we find that inhibitory G␣ signaling is sufficient to account for the actions of mGluR2 in each. The fact that constitutively active G␣ i2 occludes the transient suppression of synaptic transmission by mGluR2, while enhancing LTD, suggests further that these two forms of plasticity are expressed via different mechanisms. In addition, the LTP deficit observed in constitutively active G␣ i2-expressing mice suggests that mGluR2 activation may serve as a metaplastic switch to permit the induction of LTD by inhibiting LTP.O ne important mechanism whereby neuronal activity is controlled is through the action of presynaptic inhibitory G protein-coupled receptors (1, 2). One member of this group is the metabotropic glutamate receptor, mGluR2, which is present at mossy fiber-CA3 pyramidal cell synapses. Here, mGluR2s are localized perisynaptically near presynaptic terminals (3, 4), where they are thought to function as autoreceptors to suppress transmission in response to excess glutamate release. Pharmacological and genetic manipulations have shown that mGluR2s participate in at least three types of synaptic plasticity: (i) a transient suppression of synaptic transmission that occurs in response to receptor activation, (ii) a persistent form of synaptic depression requiring both mGluR2 activation and presynaptic calcium influx (long-term depression, LTD), and (iii) mGluR2-mediated inhibition of long-term potentiation (LTP) (5, 6).The relationship among these forms of mossy fiber-CA3 synaptic plasticity and the mechanism of mGluR2 involvement in each is not completely clear. Does the involvement of mGluR2 in both transient and persistent synaptic depression reflect a common mechanism, such that the persistent form merely results from a stabilization of the transient form, or does mGluR2 participate in these two processes via independent mechanisms? Similarly, does the inhibition of LTP by group II mGluR activation reflect opposing actions on a common effector pathway for depression and potentiation, or a permissive role for mGluR2 in inducing LTD by blocking LTP? We have begun to address these questions by exploring the signaling pathway downstream of mGluR2 using transgenic mice that express a constitutively active form of G␣ i2 . We find that the effects of mGluR2 on mossy fiber synaptic plasticity can be accounted for by the actions of inhibitory G␣ subunits. However,...

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“…GAIP, a G␣-interacting protein and a member of the RGS family of GTPaseactivating proteins (reviewed in Ref. 28), was first identified in this way; and recently two more putative ␣ targets, nucleobindin (29) and a novel LGN repeat protein (30), were described by Insel and co-workers. So far, no physiological role of the latter two G␣ targets has been determined.…”

Section: Novel ␣ Targetsmentioning

confidence: 99%

The Many Faces of G Protein Signaling

Hamm

1998

Journal of Biological Chemistry

1,044

842

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A large number of hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli exert their effects on cells and organisms by binding to G protein-coupled receptors. More than a thousand such receptors are known, and more are being discovered all the time. Heterotrimeric G proteins transduce ligand binding to these receptors into intracellular responses, which underlie physiological responses of tissues and organisms. G proteins play important roles in determining the specificity and temporal characteristics of the cellular responses to signals. They are made up of ␣, ␤, and ␥ subunits, and although there are many gene products encoding each subunit (20 ␣, 6 ␤, and 12 ␥ gene products are known), four main classes of G proteins can be distinguished: G s , which activates adenylyl cyclase; G i , which inhibits adenylyl cyclase; G q , which activates phospholipase C; and G 12 and G 13 , of unknown function.G proteins are inactive in the GDP-bound, heterotrimeric state and are activated by receptor-catalyzed guanine nucleotide exchange resulting in GTP binding to the ␣ subunit. GTP binding leads to dissociation of G␣⅐GTP from G␤␥ subunits and activation of downstream effectors by both G␣⅐GTP and free G␤␥ subunits. G protein deactivation is rate-limiting for turnoff of the cellular response and occurs when the G␣ subunit hydrolyzes GTP to GDP. The recent resolution of crystal structures of heterotrimeric G proteins in inactive and active conformations provides a structural framework for understanding their role as conformational switches in signaling pathways. As more and more novel pathways that use G proteins emerge, recognition of the diversity of regulatory mechanisms of G protein signaling is also increasing. The recent progress in the structure, mechanisms, and regulation of G protein signaling pathways is the subject of this review. Because of space considerations, I will concentrate mainly on recent studies; readers are directed to a number of excellent reviews that cover earlier studies. G Protein StructureG␣ subunits contain two domains, a domain involved in binding and hydrolyzing GTP (the G domain) that is structurally identical to the superfamily of GTPases including small G proteins and elongation factors (1) and a unique helical domain that buries the GTP in the core of the protein (2, 3) (Fig. 1). The ␤ subunit of heterotrimeric G proteins has a 7-membered ␤-propeller structure based on its 7 WD-40 repeats (4 -6). The ␥ subunit interacts with ␤ through an N-terminal coiled coil and then all along the base of ␤, making extensive contacts (Fig. 1). The ␤ and ␥ subunits form a functional unit that is not dissociable except by denaturation. G protein activation by receptors leads to GTP binding on the G␣ subunit. The structural nature of the GTP-mediated switch on the G␣ subunit is a change in conformation of three flexible regions designated Switches I, II, and III to a well ordered, GTP-bound activated conformation with lowered affinity for G␤␥ (7) (Fig. 1). This leads to increased affin...

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Interaction of the protein nucleobindin with G_αi2, as revealed by the yeast two‐hybrid system

Cited by 45 publications

References 26 publications

Calnuc, an EF-Hand Ca ²⁺ binding protein, specifically interacts with the C-terminal α5-helix of Gαi3

Calnuc, an EF-Hand Ca ²⁺ binding protein, specifically interacts with the C-terminal α5-helix of Gαi3

mGluR2 acts through inhibitory Gα subunits to regulate transmission and long-term plasticity at hippocampal mossy fiber-CA3 synapses

The Many Faces of G Protein Signaling

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Interaction of the protein nucleobindin with Gαi2, as revealed by the yeast two‐hybrid system

Cited by 45 publications

References 26 publications

Calnuc, an EF-Hand Ca 2+ binding protein, specifically interacts with the C-terminal α5-helix of Gαi3

Calnuc, an EF-Hand Ca 2+ binding protein, specifically interacts with the C-terminal α5-helix of Gαi3

mGluR2 acts through inhibitory Gα subunits to regulate transmission and long-term plasticity at hippocampal mossy fiber-CA3 synapses

The Many Faces of G Protein Signaling

Contact Info

Product

Resources

About

Interaction of the protein nucleobindin with G_αi2, as revealed by the yeast two‐hybrid system

Calnuc, an EF-Hand Ca ²⁺ binding protein, specifically interacts with the C-terminal α5-helix of Gαi3

Calnuc, an EF-Hand Ca ²⁺ binding protein, specifically interacts with the C-terminal α5-helix of Gαi3