Biomedical subjects

Randy A Hall

Publications and source records attributed to Randy A Hall.

36 records · Page 2Linked to original sources

PubMed2004

An electron microscope immunocytochemical study of GABA(B) R2 receptors in the monkey basal ganglia: a comparative analysis with GABA(B) R1 receptor distribution.

Functional gamma-aminobutyric acid (GABA)(B) receptors are heterodimers made up of GABA(B) R1 and GABA(B) R2 subunits. The subcellular localization of GABA(B) R2 receptors remains poorly known in the central nervous system. Therefore, we performed an ultrastructural analysis of the localization of GABA(B) R2 receptor immunoreactivity in the monkey basal ganglia. Furthermore, to characterize better the neuronal sites at which GABA(B) R1 and GABA(B) R2 may interact to form functional receptors, we compared the relative distribution of immunoreactivity of the two GABA(B) receptors in various basal ganglia nuclei. Light to moderate GABA(B) R2 immunoreactivity was found in cell bodies and neuropil elements in all basal ganglia nuclei. At the electron microscope level, GABA(B) R2 immunoreactivity was commonly expressed postsynaptically, although immunoreactive preterminal axonal segments were also frequently encountered, particularly in the globus pallidus and substantia nigra, where they accounted for the third of the total number of GABA(B) R2-containing elements. A few labeled terminals that displayed the ultrastructural features of glutamatergic boutons were occasionally found in most basal ganglia nuclei, except for the subthalamic nucleus, which was devoid of GABA(B) R2-immunoreactive boutons. The relative distribution of GABA(B) R2 immunoreactivity in the monkey basal ganglia was largely consistent with that of GABA(B) R1, but some exceptions were found, most noticeably in the globus pallidus and substantia nigra, which contained a significantly larger proportion of presynaptic elements labeled for GABA(B) R1 than GABA(B) R2. These findings suggest the possible coexistence and heterodimerization of GABA(B) R1 and GABA(B) R2 at various pre- and postsynaptic sites, but also raise the possibility that the formation of functional GABA(B) receptors in specific compartments of basal ganglia neurons relies on mechanisms other than GABA(B) R1/R2 heterodimerization.

Animals↗

PubMed2004

Agonist-induced polarized trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role of SNARE proteins.

Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study, we examined domain specificity of recruitment and the role of soluble N-ethylmaleimide (NEM) attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line T84 was used because it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation, and electron microscopic studies showed that the A2b receptor is intracellular at rest and that apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing vesicle-associated membrane protein (VAMP)-2. Furthermore, in cells stimulated with apical or basolateral adenosine, we demonstrate a complex consisting of VAMP-2, soluble NEM-sensitive factor attachment protein (SNAP)-23, and A2b receptor that is coimmunoprecipitated in cells stimulated with adenosine within 5 min and is no longer detected within 15 min. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98 and 90%, respectively. cAMP synthesis induced by foskolin was not affected, suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that 1) the A2b receptor is intracellular at rest; 2) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane; 3) the SNARE proteins, VAMP-2 and SNAP-23, participate in the recruitment of the A2b receptor; and 4) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.

Adenosine↗

PubMed2004

Subcellular and subsynaptic localization of group I metabotropic glutamate receptors in the monkey subthalamic nucleus.

Both subtypes of group I metabotropic glutamate receptor, mGluR1 and mGluR5, are expressed postsynaptically in neurons of the subthalamic nucleus (STN), and their activation induces different physiological responses. To test whether these effects could be explained by a differential localization of the two group I mGluRs, we analyzed the subcellular and subsynaptic distribution of mGluR1a and mGluR5 in the monkey STN. Double-immunofluorescence and light microscopic analyses revealed that both group I mGluR subtypes were strongly coexpressed in the neuropil and neuronal perikarya. Astrocytic perikarya exhibited intense mGluR1a, but no detectable mGluR5, immunoreactivity. At the electron microscopic level, immunoperoxidase labeling for both mGluR1a and mGluR5 was localized mainly in dendrites. A significant proportion of the total pool of mGluR1a-immunoreactive elements was accounted for by glial cell processes, whereas glial cell labeling was much less frequently encountered in sections immunostained for mGluR5. Preembedding immunogold labeling in STN dendrites revealed that 60-70% of the gold labeling for both mGluR subtypes was intracellular, whereas 30-40% was apposed to the plasma membrane. Of the plasma membrane-apposed particles, more than 90% were extrasynaptic; fewer than 10% were associated with symmetric or asymmetric synapses. Most of the synapse-associated labeling was found at the edges of both asymmetric and symmetric postsynaptic specializations. Some extrasynaptic gold particles were aggregated on parts of the plasma membrane tightly apposed by glial processes. These findings demonstrate that mGluR1a and mGluR5 exhibit a similar pattern of subsynaptic localization in monkey STN neurons, with both receptor subtypes exhibiting substantial extrasynaptic and perisynaptic localization.

Animals↗

PubMed2004

kappa Opioid receptor interacts with Na(+)/H(+)-exchanger regulatory factor-1/Ezrin-radixin-moesin-binding phosphoprotein-50 (NHERF-1/EBP50) to stimulate Na(+)/H(+) exchange independent of G(i)/G(o) proteins.

We previously showed that Na(+)/H(+)-exchanger regulatory factor-1/Ezrin-radixin-moesin-binding phosphoprotein-50 (NHERF-1/EBP50) co-immunoprecipitated with the human kappa opioid receptor (hKOR) and that its overexpression blocked the kappa agonist U50,488H-induced hKOR down-regulation by enhancing recycling. Here, we show that glutathione S-transferase (GST)-hKOR C-tail interacted with purified NHERF-1/EBP50, whereas GST or GST-C-tails of micro or delta opioid receptors did not. GST-hKOR C-tail, but not GST, bound HA-NHERF-1/EBP50 transfected into Chinese hamster ovary cells and endogenous NHERF-1/EBP50 in opossum kidney proximal tubule epithelial cells (OK cells). The PDZ domain I, but not II, of NHERF-1/EBP50 was involved in the interaction. Association of NHERF-1/EBP50 with hKOR C-tail enhanced oligomerization of NHERF-1/EBP50. NHERF-1/EBP50 was previously shown to regulate Na(+)/H(+)-exchanger 3 (NHE3) activities in OK cells. We found stimulation of OK cells with U50,488H significantly enhanced Na(+)/H(+) exchange, which was blocked by naloxone but not by pertussis toxin pretreatment, indicating it is mediated by KORs but independent of G(i)/G(o) proteins. In OKH cells, a subclone of OK cells expressing a much lower level of NHERF-1/EBP50, U50,488H had no effect on Na(+)/H(+) exchange, although it enhanced p44/42 mitogen-activated protein kinase phosphorylation via G(i)/G(o) proteins similar to that in OK cells. Stable transfection of NHERF-1/EBP50 into OKH cells restored the stimulatory effect of U50,488H upon Na(+)/H(+) exchange. Thus, NHERF-1/EBP50 binds directly to KOR, and this association plays an important role in accelerating Na(+)/H(+) exchange. We hypothesize that binding of the KOR to NHERF-1/EBP50 facilitates oligomerization of NHERF-1/EBP50, leading to stimulation of NHE3. This study provides the first direct evidence that a G protein-coupled receptor through association with NHERF-1/EBP-50 stimulates NHE3.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed2004

Hetero-oligomerization between GABAA and GABAB receptors regulates GABAB receptor trafficking.

The neurotransmitter gamma-aminobutyric acid (GABA) mediates inhibitory signaling in the brain via stimulation of both GABA(A) receptors (GABA(A)R), which are chloride-permeant ion channels, and GABA(B) receptors (GABA(B)R), which signal through coupling to G proteins. Here we report physical interactions between these two different classes of GABA receptor. Association of the GABA(B) receptor 1 (GABA(B)R1) with the GABA(A) receptor gamma2S subunit robustly promotes cell surface expression of GABA(B)R1 in the absence of GABA(B)R2, a closely related GABA(B) receptor that is usually required for efficient trafficking of GABA(B)R1 to the cell surface. The GABA(B)R1/gamma2S complex is not detectably functional when expressed alone, as assessed in both ERK activation assays and physiological analyses in oocytes. However, the gamma2S subunit associates not only with GABA(B)R1 alone but also with the functional GABA(B)R1/GABA(B)R2 heterodimer to markedly enhance GABA(B) receptor internalization in response to agonist stimulation. These findings reveal that the GABA(B)R1/gamma2S interaction results in the regulation of multiple aspects of GABA(B) receptor trafficking, allowing for cross-talk between these two distinct classes of GABA receptor.

Binding Sites↗

PubMed2004

Cell surface expression of alpha1D-adrenergic receptors is controlled by heterodimerization with alpha1B-adrenergic receptors.

alpha(1)-Adrenergic receptors (ARs) belong to the large Class I G protein-coupled receptor superfamily and comprise three subtypes (alpha(1A), alpha(1B), and alpha(1D)). Previous work with heterologously expressed C-terminal green fluorescent protein (GFP)-tagged alpha(1)-ARs showed that alpha(1A)- and alpha(1B)-ARs localize to the plasma membrane, whereas alpha(1D)-ARs accumulate intracellularly. We recently showed that alpha(1D)- and alpha(1B)-ARs form heterodimers, whereas alpha(1D)- and alpha(1A)-ARs do not. Here, we examined the role of heterodimerization in regulating alpha(1D)-AR localization using both confocal imaging of GFP- or CFP-tagged alpha(1)-ARs and a luminometer-based surface expression assay in HEK293 cells. Co-expression with alpha(1B)-ARs caused alpha(1D)-ARs to quantitatively translocate to the cell surface, but co-expression with alpha(1A)-ARs did not. Truncation of the alpha(1B)-AR extracellular N terminus or intracellular C terminus had no effect on surface expression of alpha(1D)-ARs, suggesting primary involvement of the hydrophobic core. Co-transfection with an uncoupled mutant alpha(1B)-AR (Delta12alpha(1B)) increased both alpha(1D)-AR surface expression and coupling to norepinephrine-stimulated Ca(2+) mobilization. Finally, GFP-tagged alpha(1D)-ARs were not detected on the cell surface when expressed in rat aortic smooth muscle cells that express no endogenous ARs, but were almost exclusively localized on the surface when expressed in DDT(1)MF-2 cells, which express endogenous alpha(1B)-ARs. These studies demonstrate that alpha(1B)/alpha(1D)-AR heterodimerization controls surface expression and functional coupling of alpha(1D)-ARs, the N- and C-terminal domains are not involved in this interaction, and that alpha(1B)-AR G protein coupling is not required. These observations may be relevant to many other Class I G protein-coupled receptors, where the functional consequences of heterodimerization are still poorly understood.

Amino Acid Sequence↗

PubMed2004

Beta-adrenergic receptors and their interacting proteins.

The three subtypes of beta-adrenergic receptor (beta AR) all interact with G proteins as a central aspect of their signaling. The various beta AR subtypes also associate differentially with a variety of other cytoplasmic and transmembrane proteins. These beta AR-interacting proteins play distinct roles in the regulation of receptor signaling and trafficking. The specificity of beta AR associations with various binding partners can help to explain key physiological differences between beta AR subtypes. Moreover, the differential tissue expression patterns of many of the beta AR-interacting proteins may contribute to tissue-specific regulation of beta AR function.

Animals↗

PubMed2004

Olfactory receptor localization and function: an emerging role for GPCR heterodimerization.

Research on olfaction has been fraught with considerable frustration because none of the hundreds of olfactory receptors make it to the cell surface on their own when expressed in heterologous systems. Recent work indicates that the heterodimerization of olfactory receptors with beta2-adrenergic receptors results in surface expression of these G protein-coupled receptors. Similar conclusions--that heterodimerization is essential for surface expression of olfactory receptors--have been drawn from research in Drosophila utilizing completely different knockout and functional approaches. Together these findings may unlock the solution to a problem that has plagued the molecular study of olfaction since the cloning of the first olfactory G protein-coupled receptor over twelve years ago.

Animals↗

PubMed2004

Studying protein-protein interactions via blot overlay or Far Western blot.

Blot overlay is a useful method for studying protein-protein interactions. This technique involves fractionating proteins on SDS-PAGE, blotting to nitrocellulose or PVDF membrane, and then incubating with a probe of interest. The probe is typically a protein that is radiolabeled, biotinylated, or simply visualized with a specific antibody. When the probe is visualized via antibody detection, this technique is often referred to as "Far Western blot." Many different kinds of protein-protein interactions can be studied via blot overlay, and the method is applicable to screens for unknown protein-protein interactions as well as to the detailed characterization of known interactions.

Animals↗

PubMed2003

Cell cycle-dependent phosphorylation of Disabled-2 by cdc2.

Disabled-2 (Dab2; also known as p96 and DOC-2) is a signal transduction protein that has been implicated in the control of cell growth. Dab2 is known to be a phosphoprotein, but little is known about the kinases that phosphorylate Dab2. We have found that Dab2 phosphorylation is markedly increased during the mitosis phase of the cell cycle. This phosphorylation is blocked by roscovitine, a selective inhibitor of cyclin-dependent kinases. Dab2 robustly coimmunoprecipitates from cells with the cyclin-dependent kinase cdc2, and purified cdc2 can phosphorylate purified Dab2 fusion proteins in vitro on multiple sites. Cellular phosphorylation of Dab2 by cdc2 promotes the association of Dab2 with Pin1, a peptidylprolyl isomerase that regulates the rate of Dab2 dephosphorylation. These findings reveal that Dab2 is differentially phosphorylated during the cell cycle by cdc2 and provide a potential feedback mechanism by which Dab2 inhibition of cell growth and proliferation may be regulated.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Heterodimerization of alpha 2A- and beta 1-adrenergic receptors.

beta- and alpha(2)-adrenergic receptors are known to exhibit substantial cross-talk and mutual regulation in tissues where they are expressed together. We have found that the beta(1)-adrenergic receptor (beta(1)AR) and alpha(2A)-adrenergic receptor (alpha(2A)AR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged beta(1)AR and alpha(2A)AR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of alpha(2A)AR was found to induce substantial internalization of coexpressed beta(1)AR, providing further evidence for a physical association between the two receptors in a cellular environment. Ligand binding assays examining displacement of [(3)H]dihydroalprenolol binding to the beta(1)AR by various ligands revealed that beta(1)AR pharmacological properties were significantly altered when the receptor was coexpressed with alpha(2A)AR. Finally, beta(1)AR/alpha(2A)AR heterodimerization was found to be markedly enhanced by a beta(1)AR point mutation (N15A) that blocks N-linked glycosylation of the beta(1)AR as well as by point mutations (N10A/N14A) that block N-linked glycosylation of the alpha(2A)AR. These data reveal an interaction between beta(1)AR and alpha(2A)AR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors.

Cells, Cultured↗

PubMed2003

Subtype-specific dimerization of alpha 1-adrenoceptors: effects on receptor expression and pharmacological properties.

The potential role of dimerization in controlling the expression and pharmacological properties of alpha1-adrenoceptor subtypes was examined using coimmunoprecipitation of epitope-tagged receptors. Human alpha1-adrenoceptor subtypes (alpha1A, alpha1B, alpha1D) were tagged at their amino-termini with Flag or hemagglutinin epitopes and transfected into human embryonic kidney 293 cells. Homodimerization of all three subtypes was observed by coimmunoprecipitation of receptors with different tags and was not altered by norepinephrine treatment. Heterodimer formation between hemagglutinin-tagged alpha1B-adrenoceptors and Flag-tagged alpha1A- or alpha1D-adrenoceptors was also observed. However, no alpha1A/alpha1D-adrenoceptor heterodimers were observed, suggesting that dimerization is subtype-specific. The extent of heterodimerization was also unaltered by norepinephrine treatment. alpha1-Adrenoceptor truncation mutants lacking carboxyl or amino-terminal sequences formed homo- and heterodimers similarly to full-length receptors, suggesting that these domains play little or no role in dimerization. Biotinylation with a membrane-impermeable agent showed that monomers and homo- and hetero-oligomers of all three subtypes are expressed on the cell surface. Radioligand binding studies showed that heterodimerization did not alter the affinity of alpha1-adrenoceptors for norepinephrine, prazosin, or subtype-selective antagonists, suggesting that dimerization does not result in pharmacologically distinct subtypes. However, coexpression of alpha1B-adrenoceptors significantly increased both binding site density and protein expression of alpha1A- and alpha1D-adrenoceptors, and increased cell surface expression of alpha1D-adrenoceptors, suggesting a functional role for heterodimerization. Conversely, coexpression of alpha1A-with alpha1D-adrenoceptors, which did not heterodimerize, had no effect on receptor density or protein. These studies demonstrate subtype-selective heterodimerization of alpha1-adrenoceptors, which does not change their pharmacological properties but seems to have functional consequences in regulating receptor expression and trafficking.

Adrenergic alpha-Antagonists↗

PubMed2002

Regulation of G protein-coupled receptor signaling by scaffold proteins.

The actions of many hormones and neurotransmitters are mediated through stimulation of G protein-coupled receptors. A primary mechanism by which these receptors exert effects inside the cell is by association with heterotrimeric G proteins, which can activate a wide variety of cellular enzymes and ion channels. G protein-coupled receptors can also interact with a number of cytoplasmic scaffold proteins, which can link the receptors to various signaling intermediates and intracellular effectors. The multicomponent nature of G protein-coupled receptor signaling pathways makes them ideally suited for regulation by scaffold proteins. This review focuses on several specific examples of G protein-coupled receptor-associated scaffolds and the roles they may play in organizing receptor-initiated signaling pathways in the cardiovascular system and other tissues.

Animals↗

PubMed2002

Glycosylation of beta(1)-adrenergic receptors regulates receptor surface expression and dimerization.

The beta(1)-adrenergic receptor (beta(1)AR) has one predicted site of N-linked glycosylation on its extracellular amino-terminus, but the glycosylation and potential functional importance of this site have not yet been examined. We show here that the beta(1)AR is glycosylated in various cell types and that mutation of the single predicted site of N-linked glycosylation (N15A) results in the formation of receptors that are not N-glycosylated. The beta(1)AR N15A mutant exhibited significantly decreased basal surface expression relative to the wild-type receptor but had no detectable deficits in ligand binding or agonist-promoted internalization. Co-immunoprecipitation experiments using Flag-tagged and HA-tagged receptors demonstrated that the beta(1)AR-N15A mutant receptor exhibits a markedly reduced capacity for dimerization relative to wild-type beta(1)AR. These data reveal that the beta(1)AR is glycosylated on Asn15 and that this glycosylation plays a role in regulating beta(1)AR surface expression and dimerization.

Amino Acid Substitution↗

PubMed2002

Novel interaction between the M4 muscarinic acetylcholine receptor and elongation factor 1A2.

The activation of the muscarinic acetylcholine receptor (mAChR) family, consisting of five subtypes (M1-M5), produces a variety of physiological effects throughout the central nervous system. However, the role of each individual subtype remains poorly understood. To further elucidate signal transduction pathways for specific subtypes, we used the most divergent portion of the subtypes, the intracellular third (i3) loop, as bait to identify interacting proteins. Using a brain pull-down assay, we identify elongation factor 1A2 (eEF1A2) as a specific binding partner to the i3 loop of M4, and not to M1 or M2. In addition, we demonstrate a direct interaction between these proteins. In the rat striatum, the M4 mAChR colocalizes with eEF1A2 in the soma and neuropil. In PC12 cells, endogenous eEF1A2 co-immunoprecipitates with the endogenous M4 mAChR, but not with the endogenous M1 mAChR. In our in vitro model, M4 dramatically accelerates nucleotide exchange of eEF1A2, a GTP-binding protein. This indicates the M4 mAChR is a guanine exchange factor for eEF1A2. eEF1A2 is an essential GTP-binding protein for protein synthesis. Thus, our data suggest a novel role for M4 in the regulation of protein synthesis through its interaction with eEF1A2.

Amino Acid Sequence↗

PubMed2002

Identification of novel G protein-coupled receptor-interacting proteins.

Biochemical and genetic methods utilizing G protein-coupled receptor fragments have been used successfully to identify G protein-coupled receptor-interacting proteins. As noted earlier, these methods may be unable to detect interactions that require certain conformations of the native receptor protein, but have nevertheless proven quite useful in expanding our understanding of receptor regulation to include interactions with proteins other than G proteins, G protein-coupled receptor kinases, and arrestins. Undoubtedly, it is likely that all G protein-coupled receptors have their own unique constellations of associated cytoplasmic proteins, and the techniques described here should prove useful in identifying these.

Animals↗

PubMed2002

Interaction between metabotropic glutamate receptor 7 and alpha tubulin.

Metabotropic glutamate receptors (mGluRs) mediate a variety of responses to glutamate in the central nervous system. A primary role for group-III mGluRs is to inhibit neurotransmitter release from presynaptic terminals, but the molecular mechanisms that regulate presynaptic trafficking and activity of group-III mGluRs are not well understood. Here, we describe the interaction of mGluR7, a group-III mGluR and presynaptic autoreceptor, with the cytoskeletal protein, alpha tubulin. The mGluR7 carboxy terminal (CT) region was expressed as a GST fusion protein and incubated with rat brain extract to purify potential mGluR7-interacting proteins. These studies yielded a single prominent mGluR7 CT-associated protein of 55 kDa, which subsequent microsequencing analysis revealed to be alpha tubulin. Coimmunoprecipitation assays confirmed that full-length mGluR7 and alpha tubulin interact in rat brain as well as in BHK cells stably expressing mGluR7a, a splice variant of mGluR7. In addition, protein overlay experiments showed that the CT domain of mGluR7a binds specifically to purified tubulin and calmodulin, but not to bovine serum albumin. Further pull-down studies revealed that another splice variant mGluR7b also interacts with alpha tubulin, indicating that the binding region is not localized to the splice-variant regions of either mGluR7a (900-915) or mGluR7b (900-923). Indeed, deletion mutagenesis experiments revealed that the alpha tubulin-binding site is located within amino acids 873-892 of the mGluR7 CT domain, a region known to be important for regulation of mGluR7 trafficking. Interestingly, activation of mGluR7a in cells results in an immediate and significant decrease in alpha tubulin binding. These data suggest that the mGluR7/alpha tubulin interaction may provide a mechanism to control access of the CT domain to regulatory molecules, or alternatively, that this interaction may lead to morphological changes in the presynaptic membrane in response to receptor activation.

Amino Acid Sequence↗

PubMedDate not supplied

Cocaine- and amphetamine-regulated transcript (CART) peptide activates the extracellular signal-regulated kinase (ERK) pathway in AtT20 cells via putative G-protein coupled receptors.

CART peptides are important neurotransmitters, but little is known about their receptors or signaling pathways in cells. In this study we describe the effects of CART 55-102 on the stimulation of extracellular signal-related kinase (ERK) in a pituitary-derived cell line. CART 55-102 treatment resulted in markedly enhanced ERK phosphorylation in AtT20 and GH3 cells, but had no significant effect on ERK phosphorylation levels in a variety of other cell types that were examined. The peptide activated ERK1 and 2 in AtT20 cells in a dose- and time-dependent manner, but an inactive peptide, CART 1-27, had no effect. U0126, an inhibitor of the MEK kinases, blocked the CART-stimulated activation of ERKs. ERK activation was also attenuated by pertussis toxin pre-treatment, but not by genistein, suggesting a Gi/o-dependent mechanism. Overall, these data strongly support the existence of a specific receptor for CART peptide that is a G-protein coupled receptor utilizing a Gi/o mechanism involving MEK1 and 2.

Analysis of Variance↗