Antagonist minigenes identify genes regulated by parathyroid hormone through G protein-selective and G protein co-regulated mechanisms in osteoblastic cells

Parathyroid hormone (PTH) is the major hormone regulating bone remodeling. Binding of PTH to the PTH1 receptor (PTH1R), a heterotrimeric G protein coupled receptor (GPCR), can potentially trigger multiple signal transduction pathways mediated through several different G proteins. In this study, we employed G protein antagonist minigenes inhibiting Gα_s, Gα_q or Gα₁₂ to selectively dissect out which of these G proteins were responsible for effects of PTH(1-34) in targeted signaling and osteogenesis arrays consisting of 159 genes. Among the 32 genes significantly regulated by 24 h PTH treatment in UMR-106 osteoblastic cells, 9 genes were exclusively regulated through G_s, 6 genes were solely mediated through G_q, and 3 genes were only controlled through G₁₂. Such findings support the concept that there is some absolute specificity in downstream responses initiated at the G protein level following binding of PTH to the PTH1R. On the other hand, 6 PTH-regulated genes were regulated by both G_s and G_q, 3 genes were regulated by both G_s and G₁₂, and 3 genes were controlled by G_s, G_q and G₁₂. These findings indicate potential overlapping or sequential interactions among different G protein-mediated pathways. In addition, two PTH-regulated genes were not regulated through any of the G proteins examined, suggesting that additional signaling mechanisms may be involved. Selectivity was largely maintained over a 2-48-hour time period. The minigene effects were mimicked by downstream inhibitors. The dissection of the differential effects of multiple G protein pathways on gene regulation provides a more complete understanding of PTH signaling in osteoblastic cells.     

Two isoforms of eukaryotic phospholipase C in Paramecium affecting transport and release of GPI-anchored proteins in vivo

Surface proteins anchored by a glycosylphosphatidylinositol (GPI) residue in the cell membrane are widely distributed among eukaryotic cells. The GPI anchor is cleavable by a phospholipase C (PLC) leading to the release of such surface proteins, and this process is postulated to be essential in several systems. For higher eukaryotes, the responsible enzymes have not been characterized in any detail as yet. Here we characterize six PLCs in the ciliated protozoan, Paramecium, which, in terms of catalytic domains and architecture, all show characteristics of PLCs involved in signal transduction in higher eukaryotes. We show that some of these endogenous PLCs can release GPI-anchored surface proteins in vitro: using RNA_i to reduce PLC expression results in the same effects as the application of PLC inhibitors. With two enzymes, PLC2 and PLC6, RNA_i phenotypes show strong defects in release of GPI-anchored surface proteins in vivo. Moreover, these RNA_i lines also show abnormal surface protein distribution, suggesting that GPI cleavage may influence trafficking of anchored proteins. As we find GFP fusion proteins in the cytosol and in the surface protein extracts, these PLCs obviously show unconventional translocation mechanisms. This is the first molecular data on endogenous Paramecium PLCs with the described properties affecting GPI anchors in vitro and in vivo.     

A role for G proteins in plant hormone signalling?

The G protein signalling pathway is one of the most highly conserved mechanisms that enables cells to sense and respond to changes in their environment. Essential components of this are cell surface G protein-coupled receptors (GPCRs) that perceive extracellular ligands, and heterotrimeric G proteins (G proteins) that transduce information from activated GPCRs to down-stream effectors such as enzymes or ion channels. It is now clear from a range of biochemical and molecular studies that some potential G protein signalling components exist in plants. The best examples of these are the seven transmembrane receptor homologue GCR1 and the G_α (GPA1) and G_β (Gβ1) subunit homologues of heterotrimeric G proteins. G protein agonists and antagonists are known to influence a variety of signalling events in plants and have been used to implicate G proteins in a range of signalling pathways that include the plant hormones gibberellin and auxin. Furthermore, antisense suppression of GCR1 expression in Arabidopsis leads to a phenotype that supports a role for this receptor in cytokinin signalling. This review considers the current evidence for and against functional G protein signalling pathways in higher plants and questions whether or not these might be involved in the action of certain plant hormones.     

CB(1) cannabinoid receptor-G protein association: a possible mechanism for differential signaling

Effects of cannabinoid compounds on neurons are predominantly mediated by the CB(1) cannabinoid receptor. Onset of signaling cascades in response to cannabimimetic drugs is triggered by the interaction of the cannabinoid receptor with G(i/o) proteins. Much work has been done to delineate the cannabinoid agonist-induced downstream signaling events; however, it remains to define the molecular basis of cannabinoid receptor-G protein interactions that stimulate these signaling pathways. In this review, we discuss several signal transduction pathways, focusing on studies that demonstrate the efficacy of CB(1) receptor agonists through G protein mediated pathways.     

Characterization of the Phosphoinositide-Linked Dopamine Receptor in a Mouse Hippocampal-Neuroblastoma Hybrid Cell Line

Abstract: Previous studies have established that dopamine (DA) can stimulate phosphoinositide (PI) metabolism in the CNS and in the periphery. The present study summarizes our attempt to find a cell line that expresses this dopaminergic system. We describe that the stable clonal HN33.11 cell line, established by fusion of mouse hippocampal cells with neuroblastoma cells (N18TG2) that originate from A/J mouse, natively expresses the D₁ DA receptor system that couples to PI hydrolysis. In this cell line, 500 µM DA or SKF38393 produced 43 and 75% increases in inositol phosphate (IP) accumulations, respectively. In contrast, noradrenaline or 5-hydroxytryptamine did not affect IP accumulations. The formation of IP that was stimulated by DA or SKF38393 was selectively blocked by the D₁ DA receptor antagonist SCH23390 with IC₅₀ values of 13 and 16 µM. This response was not mediated by the D_1A DA receptor and was cyclic AMP-independent, as HN33.11 cells did not express this receptor, and DA or SKF38393 was unable to stimulate the formation of cyclic AMP. In Ca²⁺-free/100 µM EGTA medium, basal IP level was reduced by 31.5%, but SKF38393-stimulated PI hydrolysis was not affected. SKF38393-stimulated IP accumulation was also not affected by pertussis toxin (PTX) treatment (200 ng/ml), suggesting that this dopaminergic response is mediated by PTX-insensitive G proteins. Co-immunoprecipitation studies indicated that in membranes of HN33.11 cells, D₁-like binding sites are coupled to Gα_q protein. Blockade of SKF38393-induced PI hydrolysis with antiserum against phospholipase C (PLC) isozymes, performed in permeabilized cells, as well as co-immunoprecipitation studies implicate PLCβ3 and PLCβ4 in this dopaminergically mediated PI hydrolysis cascade. The results indicate that HN33.11 cells express a D₁-like DA receptor that couples to PLCβ3/4 via Gα_q protein. These cells may therefore be a useful model system for investigating this receptor system.     

The R7 RGS Protein Family: Multi-Subunit Regulators of Neuronal G Protein Signaling

G protein-coupled receptor signaling pathways mediate the transmission of signals from the extracellular environment to the generation of cellular responses, a process that is critically important for neurons and neurotransmitter action. The ability to promptly respond to rapidly changing stimulation requires timely inactivation of G proteins, a process controlled by a family of specialized proteins known as regulators of G protein signaling (RGS). The R7 group of RGS proteins (R7 RGS) has received special attention due to their pivotal roles in the regulation of a range of crucial neuronal processes such as vision, motor control, reward behavior, and nociception in mammals. Four proteins in this group, RGS6, RGS7, RGS9, and RGS11, share a common molecular organization of three modules: (i) the catalytic RGS domain, (ii) a GGL domain that recruits Gβ₅, an outlying member of the G protein beta subunit family, and (iii) a DEP/DHEX domain that mediates interactions with the membrane anchor proteins R7BP and R9AP. As heterotrimeric complexes, R7 RGS proteins not only associate with and regulate a number of G protein signaling pathway components, but have also been found to form complexes with proteins that are not traditionally associated with G protein signaling. This review summarizes our current understanding of the biology of the R7 RGS complexes including their structure/functional organization, protein–protein interactions, and physiological roles.     

Phosphatidic acid potentiates Gαq stimulation of phospholipase C-β1 signaling

Phosphatidic acid (PA) is interactive with Gα_q-linked agonists to stimulate GPCR signaling via phospholipase C-β₁ (PLC-β₁). Phorbol 12-myristate 13-acetate (PMA) increases cellular levels of PA and phospholipase D activity (PLD). This study evaluated whether PMA can stimulate PLC-β₁ activity via PA, independent of GPCR input in transfected COS 7 cells. PMA alone had little effect on PLC activity in cells co-transfected with PLC-β₁ and Gα_q. Activated Gα_q, induced by co-transfecting muscarinic cholinergic receptor (m1R), was necessary for stimulation of PLC-β₁ activity by PMA. Stimulation by PMA was dependent on the PA-regulatory motif of PLC-β₁ implicating PA in this mechanism. PLD1 knockdown by antisense decreased responsiveness of PLC-β₁ to both PMA and carbachol. PA alone thus has little effect on PLC-β₁ activity, but PA and PLD1 synergize with activated Gα_q to stimulate PLC-β₁ signaling. Coordinate interaction with activated Gα_q may serve as an important mechanism to fine tune response to ligands while preventing spurious initiation of PLC-β signaling by PA in cells.     

Enhancement of Glucose Transport in Rat Thymocytes by Different Radical Sources

This study demonstrates that oxidative stress induced in rat thymocytes by the hydrophilic 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), the lipophilic cumene hydroperoxide (CumOOH) and the freely diffusible H₂O₂ is associated with an activation of facilitative glucose transport rate, mediated by GLUT1, the major transporter in this cell type. We compared the effects of the three tested radical sources on the kinetic transport parameters, showing that the transport rate enhancement in the treated cells can be ascribed to an increase in the V_max value, apart from the site of generation of the oxidative stress. The enhancement of glucose transport by the three oxidants in thymocytes was significantly attenuated both by protein tyrosine kinase inhibitors as genistein and tyrphostin A23 and by U73122, a phospholipase C inhibitor. Genistein and U73122 reversed also the cited increase of V_max values. It is concluded that the stimulation of glucose transport in response to different oxidants is mediated, at least in part, through reactive oxygen species (ROS)-induced stimulation of protein tyrosine kinase and phospholipase C pathways.     

Organelle motility regulated by the cell's environment: dissection of signaling pathways regulating movements of peroxisomes

Summary Chloroplasts and pigment granules are known to be intracellularly translocated upon discrete extracellular stimuli. The machineries transducing these signals inside cells are yet not understood. In studies investigating the motility of peroxisomes, we were able to identify both extracellular and intracellular signaling steps regulating movements of these organelles. Following simultaneous stimulation of CHO cells with both extracellular ATP and lysophosphatidic acid, an arrest of peroxisomes was observed. This block of motility was shown to be dependent on signaling cascades involving heterotrimeric G proteins of the class G_i/G_o, phospholipase C, calcium influx, and activation of protein kinase C as well as of mitogen-activated protein kinase. Cytosolic phospholipase A₂ is a point of convergence for these pathways, resulting in the release of arachidonic acid. This signaling pathway is specific for peroxisomes and does not influence motility of mitochondria, lysosomes, or endosomes. However, since the cytoskeleton and its associated proteins including the motor proteins play an important role in mediating motility of all cell organelles, it may well be that variant signaling cascades exist ensuring specific regulation of each distinct compartment.Abbreviations AA arachidonic acid - ATPS adenosine-5-O-(3-thiotriphosphate) - cAMP cyclic adenosine monophosphate - CaM-PK calmodulin-dependent protein kinase - CLIP cytosolic linker protein - DAG diacylglycerol - DiC₈ 1,2-dioctanoyl-sn-glycerol - GFP green-fluorescent protein - GTPS guanosine-5-O-(3-thiotriphosphate) - IP₃ inositol trisphosphate - LPA lysophosphatidic acid - MAPK mitogen-activated protein kinase - MEK MAPK kinase - PKA protein kinase A - PKC protein kinase C - cPKC classical PKC isoforms - PLA₂ phospholipase A₂ - PLAP PLA₂-activating proteinpeptide - PLC phospholipase C - PP2A protein phosphatase 2A     

Purification and characterization of G proteins from human brain: modification of GTPase activity upon phosphorylation

Summary Three G proteins from human brain membranes were purified to near homogeneity by conventional techniques including preparative electrophoresis. These G proteins were characterized by their ability to bind GTP, GDP and GTP analogs. Two of these proteins have molecular weights of 50,000 (G₅₀) and 36,000 (G₃₆), as determined on SDS-gels. G₃₆ was ADP-ribosylated by pertussis toxin. Thus, G₅₀ could represent a G_sα subunit, whereas G₃₆ could be G_iα or G_oα. G₅₀ was phosphorylated by cAMP dependent protein kinase and protein kinase C. G₃₆ was phosphorylated by a protein kinase independent of calcium and phospholipid, a proteolytic product of protein kinase C, analogous to protein kinase M. Phosphorylation of G₃₆ by this protein kinase induced a dramatic decrease in its GTPase activity. The third G protein, of molecular weight 22,000 probably belongs to the group of monomeric G proteins possessing functional similarities withras gene products. The regulation of G proteins involving calcium-dependent and independent pathways is delineated.     

The IGF-II receptor system: A G protein-linked mechanism

Based on the finding that stimulation of the IGF-II, receptor (IGF-IIR) is capable of activating G_i2 and calcium channels in BALB/c 3T3 fibroblasts, it was found that purified IGF-IIR can couple directly to purified G_i2 in phospholipid vesicles. IGF-IIR–G_i2 coupling can be characterized as follows. IGF-IIR directly couples to G_i2 in response to IGF-II in a stoichiometrical manner, suggesting that IGF-IIR works as a transmembrane signaling molecule and that the seven-transmembrane structure is not essential for receptor-G protein coupling. The mode of IGF-IIR–G_i2 interaction is similar to that of conventional receptor–G protein coupling, suggesting that a common G protein recognition mechanism is shared by IGF-IIR and conventional G-coupled receptors. The action of IGF-IIR is specific on G_i2 among various G proteins. Finally, the activity of IGF-IIR on G_i2 is similarly potent across the species of the proteins. These characteristics led to the discovery of a 14-amino-acid region in IGF-IIR that can directly interact with and activate G_i2, and is located at residues 2410–2423 of the human receptor. Subsequent work has indicated that this region is responsible for G_i-coupling function of intact IGF-IIR. The most important extensions of this discovery are the following: (1) The structure–function relationship for the G_i-activating function of this 14-amino-acid sequence, (2) the prediction of G protein-coupled functions of receptors based on the results obtained from 1), and (3) clarification of the detailed mechanism whereby ligand–receptor complex recognizes G proteins. This paper reviews what we have learned from IGF-IIR in terms of receptor–G protein interfaces and discusses future prospects. © 1993 Wiley-Liss, Inc.     

The ins and outs of the intermembrane space: Diverse mechanisms and evolutionary rewiring of mitochondrial protein import routes

Background

Mitochondrial biogenesis is an essential process in all eukaryotes. Import of proteins from the cytosol into mitochondria is a key step in organelle biogenesis. Recent evidence suggests that a given mitochondrial protein does not take the same import route in all organisms, suggesting that pathways of mitochondrial protein import can be rewired through evolution. Examples of this process so far involve proteins destined to the mitochondrial intermembrane space (IMS).

Scope of review

Here we review the components, substrates and energy sources of the known mechanisms of protein import into the IMS. We discuss evolutionary rewiring of the IMS import routes, focusing on the example of the lactate utilisation enzyme cytochrome b₂ (Cyb2) in the model yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans.

Major conclusions

There are multiple import pathways used for protein entry into the IMS and they form a network capable of importing a diverse range of substrates. These pathways have been rewired, possibly in response to environmental pressures, such as those found in the niches in the human body inhabited by C. albicans.

General significance

We propose that evolutionary rewiring of mitochondrial import pathways can adjust the metabolic fitness of a given species to their environmental niche. This article is part of a Special Issue entitled Frontiers of Mitochondrial.     

Agonist-Induced, GTP-Dependent Phosphoinositide Hydrolysis in Postmortem Human Brain Membranes

Abstract: Membranes prepared from postmortem human brain were used to measure the activities of three components of the phosphoinositide second messenger system. [³H] Phosphatidylinositol ([³H] PI) hydrolysis was stimulated by directly activating phospholipase C with calcium, by activating guanine nucleotide-binding proteins (G proteins) with guanosine-5′-O-(3-thiotriphosphate) (GTPγS) or with AIF₄, and by receptors activated with several agonists (in the presence of GTPγS), including (in order of increasing magnitudes of responses) carbachol, pilocarpine, histamine, trans-1-aminocyclopentyl-1, 3-dicarboxylic acid (a selective excitatory amino acid metabotropic receptor agonist), serotonin, and ATP. G_q/11 was identified as the G protein most likely to mediate [³H] PI hydrolysis in human brain membranes based on the findings that this process was not impaired by pretreatment with pertussis toxin and it was inhibited by antibodies specific for the α-subunit of G_q/11 but not by antibodies for G_o or G₁₁. The effects of postmortem delay on [³H] PI hydrolysis were examined by studying tissues obtained 6–21 h postmortem. A slight increase in basal [³H] PI hydrolysis was associated with increased postmortem time, suggesting a slow loss of the normal inhibitory control of phospholipase C. GTPγS- stimulated [³H] PI hydrolysis was unaffected by postmortem times within this range, but carbachol-induced [³H] PI hydrolysis tended to decrease with increasing postmortem times. These results demonstrate that the entire phosphoinositide complex remains functional and experimentally detectable in postmortem human brain membranes. This method provides a means to study the function, regulation, effects of diseases, and responses to drugs of the phosphoinositide system in human brain.     

Single amino acid of an octopamine receptor as a molecular switch for distinct G protein couplings

Octopamine (OA) is thought to be the invertebrate counterpart of noradrenaline and regulates various behavioral patterns of invertebrates by activating OA receptors. As a typical G protein-coupled receptor, BmOAR1, a Bombyx mori α-adrenergic-like OA receptor, is coupled to both G_s and G_q proteins to induce the release of the intracellular second messengers cAMP and Ca²⁺. In this study, we examined the pharmacological and functional properties of the cloned OA receptor, using OA enantiomers. The wild-type OA receptor exhibited significant stereoselectivity for OA enantiomers in cAMP production and binding affinity, but not in calcium signaling response. On the contrary, the Y412F mutant abolished the discrimination between OA enantiomers in the binding affinity and did not evoke any cAMP signaling response. This mutant exhibited levels of potency and efficacy similar to those of the wild-type receptor in the calcium assays. Taken together, these results suggest that Tyr412 might act as a molecular switch to regulate distinct G protein couplings, and a sequential activation model is proposed for such specific-residue-dependent, selective activation in receptors that are coupled to multiple G proteins.     

Mapping protein interactions of sodium channel NaV1.7 using epitope‐tagged gene‐targeted mice

The voltage‐gated sodium channel Na_V1.7 plays a critical role in pain pathways. We generated an epitope‐tagged Na_V1.7 mouse that showed normal pain behaviours to identify channel‐interacting proteins. Analysis of Na_V1.7 complexes affinity‐purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7 in vivo. The sodium channel β3 (Scn3b), rather than the β1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing‐response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel Na_V1.7 protein interactors including membrane‐trafficking protein synaptotagmin‐2 (Syt2), L‐type amino acid transporter 1 (Lat1) and transmembrane P24‐trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co‐immunoprecipitation (Co‐IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein‐regulated inducer of neurite outgrowth (Gprin1), an opioid receptor‐binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope‐tagged mouse should provide useful insights into the many functions now associated with the Na_V1.7 channel.     

Autophagic proteins

Oxygen (O₂), while essential for aerobic life, can also cause metabolic toxicity through the excess generation of reactive oxygen species (ROS). Pathological changes in ROS production can originate through the partial reduction of O₂ during mitochondrial electron transport, as well as from enzymatic sources. This phenomenon, termed the oxygen paradox, has been implicated in aging and disease, and is especially evident in critical care medicine. Whereas high O₂ concentrations are utilized as a life-sustaining therapeutic for respiratory insufficiency, they in turn can cause acute lung injury. Alveolar epithelial cells represent a primary target of hyperoxia-induced lung injury. Recent studies have indicated that epithelial cells exposed to high O₂ concentrations die by apoptosis, or necrosis, and can also exhibit mixed-phenotypes of cell death (aponecrosis). Autophagy, a cellular homeostatic process responsible for the lysosomal turnover of organelles and proteins, has been implicated as a general response to oxidative stress in cells and tissues. This evolutionarily conserved process is finely regulated by a complex interplay of protein factors. During autophagy, senescent organelles and cellular proteins are sequestered in autophagic vacuoles (autophagosomes) and subsequently targeted to the lysosome, where they are degraded by lysosomal hydrolases, and the breakdown products released for reutilization in anabolic pathways. Autophagy has been implicated as a cell survival mechanism during nutrient-deficiency states, and more generally, as a determinant of cell fate. However, the mechanisms by which autophagy and/or autophagic proteins potentially interact with and/or regulate cell death pathways during high oxygen stress, remain only partially understood.     

Na/H Exchanger Regulatory Factors Control Parathyroid Hormone Receptor Signaling by Facilitating Differential Activation of Gα Protein Subunits

The Na/H exchanger regulatory factors, NHERF1 and NHERF2, are adapter proteins involved in targeting and assembly of protein complexes. The parathyroid hormone receptor (PTHR) interacts with both NHERF1 and NHERF2. The NHERF proteins toggle PTHR signaling from predominantly activation of adenylyl cyclase in the absence of NHERF to principally stimulation of phospholipase C when the NHERF proteins are expressed. We hypothesized that this signaling switch occurs at the level of the G protein. We measured G protein activation by [³⁵S]GTPγS binding and Gα subtype-specific immunoprecipitation using three different cellular models of PTHR signaling. These studies revealed that PTHR interactions with NHERF1 enhance receptor-mediated stimulation of Gα_q but have no effect on stimulation of Gα_i or Gα_s. In contrast, PTHR associations with NHERF2 enhance receptor-mediated stimulation of both Gα_q and Gα_i but decrease stimulation of Gα_s. Consistent with these functional data, NHERF2 formed cellular complexes with both Gα_q and Gα_i, whereas NHERF1 was found to interact only with Gα_q. These findings demonstrate that NHERF interactions regulate PTHR signaling at the level of G proteins and that NHERF1 and NHERF2 exhibit isotype-specific effects on G protein activation.     

Yeast Ste2 receptors as tools for study of mammalian protein kinases and adaptors involved in receptor trafficking

Background

Mammalian receptors that couple to effectors via heterotrimeric G proteins (e.g., beta ₂-adrenergic receptors) and receptors with intrinsic tyrosine kinase activity (e.g., insulin and IGF-I receptors) constitute the proximal points of two dominant cell signaling pathways. Receptors coupled to G proteins can be substrates for tyrosine kinases, integrating signals from both pathways. Yeast cells, in contrast, display G protein-coupled receptors (e.g., alpha-factor pheromone receptor Ste2) that have evolved in the absence of receptor tyrosine kinases, such as those found in higher organisms. We sought to understand the motifs in G protein-coupled receptors that act as substrates for receptor tyrosine kinases and the functional consequence of such phosphorylation on receptor biology. We expressed in human HEK 293 cells yeast wild-type Ste2 as well as a Ste2 chimera engineered with cytoplasmic domains of the beta₂-adrenergic receptor and tested receptor sequestration in response to activation of the insulin receptor tyrosine kinase.

Results

The yeast Ste2 was successfully expressed in HEK 293 cells. In response to alpha-factor, Ste2 signals to the mitogen-activated protein kinase pathway and internalizes. Wash out of agonist and addition of antagonist does not lead to Ste2 recycling to the cell membrane. Internalized Ste2 is not significantly degraded. Beta₂-adrenergic receptors display internalization in response to agonist (isoproterenol), but rapidly recycle to the cell membrane following wash out of agonist and addition of antagonist. Beta₂-adrenergic receptors display internalization in response to activation of insulin receptors (i.e., cross-regulation), whereas Ste2 does not. Substitution of the cytoplasmic domains of the β₂-adrenergic receptor for those of Ste2 creates a Ste2/beta₂-adrenergic receptor chimera displaying insulin-stimulated internalization.

Conclusion

Chimera composed of yeast Ste2 into which domains of mammalian G protein-coupled receptors have been substituted, when expressed in animal cells, provide a unique tool for study of the regulation of G protein-coupled receptor trafficking by mammalian receptor tyrosine kinases and adaptor proteins.