Restricting mobility of Gsα relative to the β2-adrenoceptor enhances adenylate cyclase activity by reducing Gsα GTPase activity

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either G␣ s or G␣ q and the NK1 receptor with a truncated tail, which secured nonpromiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated G␣ s fusion construct, whereas the lower affinity component was displayed by the tail-truncated G␣ q fusion. The elusive difference between the affinity determined in heterologous versus homologous binding assays for substance P and especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from G␣ s signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated G␣ q fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the G␣ s and G␣ q species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or "signal-transductosomes" within the cell membrane.Many G-protein-coupled 7TM 1 (transmembrane segment) receptors have the capacity to interact with multiple G-proteins and thus regulate more than one effector pathway. Some receptors preferentially couple to one G-protein, but in the presence of higher, non-physiological concentrations of agonist or in, for example reconstitution assays, these receptors are able to couple to other G-proteins (1, 2). Other more promiscuous receptors couple functionally to more than one G-protein in the physiological range of agonist concentration (3-6). For example, the neurokinin NK1 receptor is coupled to two different signaling pathways: a G␣ q pathway, which activates phospholipase C ␤, thereby initiating inositol phosphate formation and a G␣ s pathway, which induces cAMP formation. Both effector systems are activated by nanomolar physiological concentrations of substance P (6). Other endogenous neurokinin peptides such as NKA and NKB as well as artificial peptide ligands such as septide can act as high affinity agonists on the NK1 receptor (7). Interestingly, in whole cell binding experiments, the NK1 receptor displays certain characteristics that indicate that the receptor in the cell membrane occurs in more than one high affinity active molecular form. First, competition binding experiments using the agonist substance P as a tracer reveal two-component binding curves for all agonists, indicating the occurrence of ...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Two Active Molecular Phenotypes of the Tachykinin NK1 Receptor Revealed by G-protein Fusions and Mutagenesis

Holst

Hastrup

Raffetseder

et al. 2001

“…There is evidence suggesting that both kinds of modification can target their cognate protein (GAP43, G i/o subunit, Lyn) to such membrane specializations (20,(43)(44)(45)(46). Theoretically, it should be possible to design tethering signals that do not target the G-protein to rafts and thus examine the consequences for signaling (47). Indeed, recently differential trafficking to membrane domains has been observed for a variety lipid modifications attached to fluorescent proteins (48).…”

Section: Fluorescent G-proteinsmentioning

confidence: 99%

A Novel Strategy to Engineer Functional Fluorescent Inhibitory G-protein α Subunits

Leaney¹,

Benians²,

Graves

et al. 2002

Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein ␣ subunits. We have designed G i/o ␣ subunits fused to the cyan fluorescent protein and assayed their function by studying the following two signal transduction pathways: the regulation of G-proteingated inwardly rectifying K ؉ channels (Kir3.0 family) and adenylate cyclase. Palmitoylation and myristoylation consensus sites were removed from G i/o ␣ subunits (G i1 ␣, G i2 ␣, G i3 ␣, and G oA ␣) and a mutation introduced at Cys ؊4 rendering the subunit resistant to pertussis toxin. This construct was fused in-frame with cyan fluorescent protein containing a short peptide motif from GAP43 that directs palmitoylation and thus membrane targeting. Western blotting confirmed G i/o ␣ protein expression. Confocal microscopy and biochemical fractionation studies revealed membrane localization. Each mutant G i/o ␣ subunit significantly reduced basal current density when transiently expressed in a stable cell line expressing Kir3.1 and Kir3.2A, consistent with the sequestration of the G␤␥ dimer by the mutant G␣ subunit. Moreover, each subunit was able to support A1-mediated and D2S-mediated channel activation when transiently expressed in pertussis toxin-treated cells. Overexpression of tagged G i3 ␣ and G oA ␣ ␣ subunits reduced receptor-mediated and forskolin-induced cAMP mobilization.

“…Following that observation, similar constructs were created for a variety of studies, as extensively reviewed in Refs. 2-4. Often the 1:1 stoichiometry, presumably imposed by fission, was reported to facilitate the study of GPCR-mediated activation of ␣-subunits in transfected cells (5)(6)(7)(8)(9)(10)(11)(12), or it was exploited to test the specificity of signals generated by GPCR and multiple ␣-subunits (13)(14)(15)(16)(17)(18)(19)(20). However, the interpretation of experiments in which fusion proteins are used as investigational probes requires an understanding of how receptor and G proteins interact in such chimeric proteins.…”

mentioning

confidence: 99%

Promiscuous Coupling at Receptor-Gα Fusion Proteins

Molinari

Ambrosio

Riitano

et al. 2003

Fusion proteins between heptahelical receptors (GPCR) and G protein ␣-subunits show enhanced signaling efficiency in transfected cells. This is believed to be the result of molecular proximity, because the interaction between linked modules of one protein chain, if not constrained by structure, should be strongly favored compared with the same in which partners react as free species. To test this assumption we made a series of fusion proteins (type 1 and 4 opioid receptors with G o and ␤ 2 adrenergic and dopamine 1 receptors with G sL ) and some mutated analogs carrying different tags and defective GPCR or G␣ subunits. Using cotransfection experiments with readout protocols able to distinguish activation at fused and non-fused ␣-subunits, we found that both the GPCR and the G␣ limb of one fusion protein can freely interact with non-fused proteins and the tethered partners of a neighboring fusion complex. Moreover, a bulky polyanionic inhibitor can suppress with identical potency receptor-G␣ interaction, either when occurring between latched domains of a fused system or separate elements of distinct molecules, indicating that the binding surfaces are equally accessible in both cases. These data demonstrate that there is no entropy drive from the linked condition of fusion proteins and suggest that their signaling may result from the GPCR of one complex interacting with the ␣-subunit of another. Moreover, the enhanced coupling efficiency commonly observed for fusion proteins is not due to the receptor tether, but to the transmembrane helix that anchors G␣ to the membrane.Fusion genes between a G protein-coupled receptor (GPCR) 1 and the corresponding G protein ␣-subunit are not found in nature, but have been generated in several laboratories over the past years. First shown by Bertin et al. (1), if a GPCR with the carboxyl terminus tied to the amino terminus of an ␣-subunit are expressed as a single polypeptide, their functional interactions and the consequent signaling activity are preserved and even enhanced. Following that observation, similar constructs were created for a variety of studies, as extensively reviewed in Refs. 2-4.Often the 1:1 stoichiometry, presumably imposed by fission, was reported to facilitate the study of GPCR-mediated activation of ␣-subunits in transfected cells (5-12), or it was exploited to test the specificity of signals generated by GPCR and multiple ␣-subunits (13-20). However, the interpretation of experiments in which fusion proteins are used as investigational probes requires an understanding of how receptor and G proteins interact in such chimeric proteins.The underlying assumption is that the proximal GPCR unit in each fused assembly can interact with the distal tethered ␣-subunit. Thus, what is normally an intermolecular binding reaction between separate partners is believed to become, by fusion, an intramolecular interaction between flanked domains of the same protein. This assumption comes from thermodynamic considerations. If the binding surfaces of the receptor and the G␣ ...