Parkinson disease is a neurodegenerative disorder whose symptoms are caused by the loss of dopaminergic neurons innervating the striatum. As striatal dopamine levels fall, striatal acetylcholine release rises, exacerbating motor symptoms. This adaptation is commonly attributed to the loss of interneuronal regulation by inhibitory D(2) dopamine receptors. Our results point to a completely different, new mechanism. After striatal dopamine depletion, D(2) dopamine receptor modulation of calcium (Ca(2+)) channels controlling vesicular acetylcholine release in interneurons was unchanged, but M(4) muscarinic autoreceptor coupling to these same channels was markedly attenuated. This adaptation was attributable to the upregulation of RGS4-an autoreceptor-associated, GTPase-accelerating protein. This specific signaling adaptation extended to a broader loss of autoreceptor control of interneuron spiking. These observations suggest that RGS4-dependent attenuation of interneuronal autoreceptor signaling is a major factor in the elevation of striatal acetylcholine release in Parkinson disease.

Abstract-Activation of the ␣ 1A -adrenergic receptor (␣ 1A -AR)/Gq pathway has been implicated as a critical trigger for the development of cardiac hypertrophy. However, direct evidence from in vivo studies is still lacking. To address this issue, transgenic mice with cardiac-targeted overexpression of the ␣ 1A -AR (4-to 170-fold) were generated, using the rodent ␣-myosin heavy chain promoter. Heterozygous animals displayed marked enhancement of cardiac contractility, evident from increases in dP/dt max (80%, PϽ0.0001), dP/dt max /LVP inst (76%, PϽ0.001), dP/dt max :dP/dt min (104%, PϽ0.0001), and fractional shortening (33%, PϽ0.05). Moreover, changes in the dP/dt max -end-diastolic volume relationship provided load-independent evidence of a primary increase in contractility. Blood pressure and heart rate were largely unchanged, and there was a small increase in (Ϫ)norepinephrine-stimulated, but not basal, phospholipase C activity. Increased contractility was directly related to the level of receptor overexpression and could be completely reversed by acute ␣ 1A -but not ␤-AR blockade. Despite the robust changes in contractility, transgenic animals displayed no morphological, histological, or echocardiographic evidence of left ventricular hypertrophy. In addition, apart from an increase in atrial natriuretic factor mRNA, expression of other hypertrophy-associated genes was unchanged. To our knowledge, these data provide the first in vivo evidence for an inotropic action of the ␣ 1A -AR.

Gα12/13 have been implicated in numerous cellular processes, however, their roles in vertebrate gastrulation are largely unknown. Here, we show that during zebrafish gastrulation, suppression of both Gα12 and Gα13 signaling by overexpressing dominant negative proteins and application of antisense morpholino-modified oligonucleotide translation interference disrupted convergence and extension without changing embryonic patterning. Analyses of mesodermal cell behaviors revealed that Gα12/13 are required for cell elongation and efficient dorsalward migration during convergence independent of noncanonical Wnt signaling. Furthermore, Gα12/13 function cell-autonomously to mediate mediolateral cell elongation underlying intercalation during notochord extension, likely acting in parallel to noncanonical Wnt signaling. These findings provide the first evidence that Gα12 and Gα13 have overlapping and essential roles in distinct cell behaviors that drive vertebrate gastrulation.

A yeast two-hybrid approach was used to discern possible new effectors for the ␤␥ subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to G␤, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, coimmunoprecipitate with G␤␥ using an anti-G␤ antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with G␤ 1 ␥ 1 but also unexpectedly with the transducin heterotrimer G␣ t ␤ 1 ␥ 1 . G␣ t alone does not interact, but it must contribute to the interaction because the apparent EC 50 value of RACK1 for G␣ t ␤ 1 ␥ 1 is 3-fold greater than that for G␤ 1 ␥ 1 (0.1 versus 0.3 M). RACK1 is a scaffold that interacts with several proteins, among which are activated ␤IIPKC and dynamin-1 (1). ␤IIPKC and dynamin-1 compete with G␤ 1 ␥ 1 and G␣ t ␤ 1 ␥ 1 for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that G␤␥ interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.Heterotrimeric G proteins are a family of proteins that transduce an extracellular signal to an intracellular response via a seven helical transmembrane G protein-coupled receptor (GPCR).1 Upon activation, the receptor facilitates the exchange of GDP for GTP in the G␣ subunit. G␣ is then thought to dissociate from the G␤␥ heterodimer allowing both complexes to individually activate a number of effectors (2, 3). Free G␤␥ interacts with a large assortment of effector proteins, including phospholipases (4), adenylyl cyclases (5), ion channels (6), and G protein-coupled receptor kinases (7). There are, however, G protein-coupled receptor responses, such as MAP kinase activation (8 -10), receptor internalization (11, 12), and organelle transport (13-15) that are mediated through the G␤␥ subunit but that have not been definitively linked to known G␤␥ effectors.G␤ is the prototypical member of a family of proteins known as WD40 repeat proteins, which seem to function as adaptors and enzyme regulators (16,17). G␤ is the only WD40 repeat protein whose three-dimensional structure is known, and it exhibits a toroidal bladed ␤-propeller structure, with each blade consisting of 4 anti-parallel ␤-strands (18). Because the WD repeat motif is a structural element of the ␤-propeller, all of these proteins are thought to be ␤-propeller proteins with a variable number of blades. Furthermore, G␤ subunits are known to interact with G␥ subunits, proteins containing a G␥-like domain (19), a pleckstrin homology domain (20), a QXXER domain (found in adenylyl cyclases) (21), and a domain contained within phosducin and its relatives (22). In this work we propose that G␤␥ also interacts with certain other WD40 repeat prote...

Epiboly spreads and thins the blastoderm over the yolk cell during zebrafish gastrulation, and involves coordinated movements of several cell layers. Although recent studies have begun to elucidate the processes that underlie these epibolic movements, the cellular and molecular mechanisms involved remain to be fully defined. Here, we show that gastrulae with altered Gα12/13 signaling display delayed epibolic movement of the deep cells, abnormal movement of dorsal forerunner cells, and dissociation of cells from the blastoderm, phenocopying e-cadherin mutants. Biochemical and genetic studies indicate that Gα12/13 regulate epiboly, in part by associating with the cytoplasmic terminus of E-cadherin, and thereby inhibiting E-cadherin activity and cell adhesion. Furthermore, we demonstrate that Gα12/13 modulate epibolic movements of the enveloping layer by regulating actin cytoskeleton organization through a RhoGEF/Rho-dependent pathway. These results provide the first in vivo evidence that Gα12/13 regulate epiboly through two distinct mechanisms: limiting E-cadherin activity and modulating the organization of the actin cytoskeleton.

Science 264, 1593-1596). Here, we evaluated the ability of G h as compared with G q to mediate receptor-stimulated inositol phosphate turnover by the three ␣ 1 -subtypes (␣ 1A , ␣ 1B , and ␣ 1D ). In addition, we questioned if the transglutaminase function of G h is involved in its receptor signaling activity. A mutant form of a human TGase II cDNA in which the codon for the active site cysteine (Cys 277 ) was replaced by serine was cloned into the mammalian expression vector pMT2. Compared with wild-type TGase II, no transglutaminase activity was observed with transient transfection of this Cys3 Ser mutant in COS-1 cells. However, like wild-type TGase, the Cys3 Ser mutant mediated receptor-stimulated inositol phosphate turnover when cotransfected with an ␣ 1B -AR cDNA. G ␣q supported ␣ 1 -AR-mediated inositol phosphate turnover by all three receptor subtypes. By contrast, although both the wild-type and Cys3 Ser construct mediated receptor signaling by the ␣ 1B AR and ␣ 1D AR, the ␣ 1A -AR was unable to interact with G h . However, a G h -dependent signaling phenotype could be rescued by a chimeric ␣ 1A construct in which the third intracellular loop of the ␣ 1A -AR was replaced by that of the ␣ 1B -AR. Thus, the signaling function of G h is independent of its transglutaminase activity and is ␣ 1 -AR subtype specific. This subtype specificity of the interaction between ␣ 1 ARs and G h involves important determinants in their third intracellular loops.

A growing body of evidence indicates that G protein-coupled receptors (GPCRs) are involved in breast tumor progression and that targeting GPCRs may be a novel adjuvant strategy in cancer treatment. However, due to the redundant role of multiple GPCRs in tumor development, it may be necessary to target a common signaling component downstream of these receptors to achieve maximum efficacy. GPCRs transmit signals through heterotrimeric G proteins composed of G␣ and G␤␥ subunits. Here we evaluated the role of G␤␥ in breast tumor growth and metastasis both in vitro and in vivo. Our data show that blocking G␤␥ signaling with G␣ t or small molecule inhibitors blocked serum-induced breast tumor cell proliferation as well as tumor cell migration induced by various GPCRs in vitro. Moreover, induced expression of G␣ t in MDA-MB-231 cells inhibited primary tumor formation and retarded growth of existing breast tumors in nude mice. Blocking G␤␥ signaling also dramatically reduced the incidence of spontaneous lung metastasis from primary tumors and decreased tumor formation in the experimental lung metastasis model. Additional studies indicate that G␤␥ signaling may also play a role in the generation of a tumor microenvironment permissive for tumor progression, because the inhibition of G␤␥ signaling attenuated leukocyte infiltration and angiogenesis in primary breast tumors. Taken together, our data demonstrate a critical role of G␤␥ signaling in promoting breast tumor growth and metastasis and suggest that targeting G␤␥ may represent a novel therapeutic approach for breast cancer.Breast cancer is the most frequently diagnosed cancer in women (1). Despite recent advances in its diagnosis and treatment with adjuvant targeted therapies, breast cancer remains the second most common cause of cancer death among women in the United States (2). As many as 90% of cancer deaths are caused by metastatic spread from primary tumors because the majority of currently marketed anticancer drugs have little effect on tumors at this stage. In light of this situation, efforts to better understand the mechanisms underlying tumor metastasis and to identify novel treatment strategies are warranted.

combined with a previously described constitutively active ␣ 1B -AR mutant, A293E, indicated that although not required for spontaneous receptor isomerization from the basal state, R, to a partially activated conformation R, interaction of Phe 310 with catecholamine agonists is essential for isomerization from R to the fully activated state, R*.1 are members of the heptahelical superfamily that share a common structural motif of seven putative ␣-helical transmembrane spanning regions linked by three extra-and three intracellular loops, an extracellular N terminus and intracellular C-terminal tail. Transmembrane signaling by all ␣ 1 -AR subtypes (␣ 1A , ␣ 1B , and ␣ 1D ) in response to the natural catecholamine agonists, norepinephrine and epinephrine, is mediated by G-proteins of the G q/11 family or in some instances, the G h family of tissue transglutaminases (1, 2). Based on certain key structural features, ␣ 1 -ARs are more closely related to rhodopsin or the type A subfamily of GPCRs that includes ␤-ARs, than to the calcitonin (type B) or metabotropic (type C) subfamilies.Binding of catecholamines by both ␣ 1 -and ␤-ARs involves the formation of a salt bridge between the basic aliphatic nitrogen atom common to all sympathomimetic amines and an aspartate (Asp 125 in the hamster ␣ 1B -AR; Asp 113 in the hamster ␤ 2 -AR) in the third transmembrane segment (TMIII) (3, 4). With rhodopsin, light induced isomerization of the retinal chromophore leads to deprotonation of a Schiff base linking the chromophore to Lys 296 in TMVII (5). In the ground state the protonated Schiff base is ionically bonded to a TMIII acidic residue (Glu 113 ) that is equivalent to Asp 125 and Asp 113 in the ␣ 1B and ␤ 2 -ARs, respectively (5). With the ␣ 1B -AR there is evidence that receptor activation also is due to disruption of an ionic interaction between the TMIII aspartate and a TMVII lysine (Lys 331 ), due to competition between the catechol protonated amine and the TMVII lysine, for binding to the TMIII aspartate (3). The TMIII aspartate thus serves as a counterion and most likely is an important residue for agonist binding and activation of all adrenergic receptors.For the ␤ 2 -AR, two serine residues, Ser 204 and Ser 207 , which are conserved in most adrenergic receptors, have been proposed to hydrogen bond with the meta-and para-hydroxyls, respectively, of the catechol ring (6). Mutation of either serine to an alanine results in a 30-fold decrease in affinity for catecholamine agonists, and each serine contributes about 50% to receptor activation. Thus, binding of both catechol hydroxyls is required for full agonist activity. By contrast, agonist binding to the ␣ 1A -AR involves an interaction between the meta-hydroxyl and Ser 188 (equivalent to Ser 203 , not Ser 204 in the ␤ 2 -AR) that plays a major role in receptor activation, being responsible for 70 -90% of the wild-type response. An interaction between the para-hydroxyl and Ser 192 (equivalent to Ser 207 in the ␤ 2 -AR), on the other hand, contributes minimally to recep...