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At least 325 records · Page 18Linked to original sources
PubMed2002

Role of the CC chemokine receptor 9/TECK interaction in apoptosis.

Chemokine receptors are members of the G protein coupled receptor (GPCR) supergene family whose expression is highly restricted to hematopoietic cells. Although the primary role of chemokine and chemokine receptor interaction is believed to be regulation of chemotaxis of leukocytes, subsequent information clearly suggests that multiple immune regulatory functions are attributed to chemokine receptor signaling. We recently showed that activation of the CC chemokine 9 receptor (CCR9), a thymus-specific chemokine receptor, led to potent cFLIP(L)-independent resistance to cycloheximide-induced apoptosis and modest resistance to Fas-mediated apoptosis possibly via activation of multiple signaling components involving Akt and glycogen synthase kinase 3beta. The fact that these two apoptotic signals involve activation of similar arrays of death execution machinery such as caspase-8, caspase-9, or caspase-3, suggests that chemokine receptor signaling may provide a wide range of antiapoptotic activities to hematopoietic cells under certain biological conditions. GPCR is a large family of cell surface receptors, many of which are critically involved in hormonal and behavioral control. Recent observations also suggest that GPCR signaling plays a pivotal role in immune cell activation. Heterotrimeric G protein is an integral part of GPCR signaling. Thus, dissection of signaling components involved in the CCR9-mediated antiapoptosis could be a framework for cell survival mechanisms and may provide options for therapeutic interventions for neurdegenerative diseases or T cell malfunctioning.

Animals↗
PubMed2001

New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades.

Activation of classical second messenger cascades cannot fully explain the recently appreciated roles of heptahelical, or G-protein coupled receptors (GPCRs), in stimulation of mitogen activated protein kinase (MAPK) cascades. Rather, several distinct signaling mechanisms appear to contribute to GPCR-mediated MAPK activation. These include transactivation of the Epidermal Growth Factor Receptor (EGFR) via the autocrine/paracrine release of EGF-like ligands at the cell surface and scaffolding of MAPK cascades. A significant advance in the understanding of how GPCRs activate MAPK cascades is the discovery that beta-arrestin, a protein well known for its roles in both receptor desensitization and internalization, serves as a scaffolding protein for at least two GPCR stimulated MAPK cascades, the extracellular signal regulated kinase (ERK) cascade and the c-jun N-terminal kinase 3 (JNK3) cascade. Together, these novel mechanisms of GPCR-mediated MAPK regulation may permit GPCRs in specific situations to control the temporal and spatial activity of MAPKs and thereby determine the consequences of GPCR stimulation with respect to transcriptional activation, cell proliferation and apoptosis.

Endocytosis↗
PubMed2001

Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C G-protein-coupled receptors.

In 1991 a new type of G-protein-coupled receptor (GPCR) was cloned, the type 1a metabotropic glutamate (mGlu) receptor, which, despite possessing the defining seven-transmembrane topology of the GPCR superfamily, bore little resemblance to the growing number of other cloned GPCRs. Subsequent studies have shown that there are eight mammalian mGlu receptors that, together with the calcium-sensing receptor, the GABA(B) receptor (where GABA is gamma-aminobutyric acid) and a subset of pheromone, olfactory and taste receptors, make up GPCR family C. Currently available data suggest that family C GPCRs share a number of structural, biochemical and regulatory characteristics, which differ markedly from those of the other GPCR families, most notably the rhodopsin/family A GPCRs that have been most widely studied to date. This review will focus on the group I mGlu receptors (mGlu1 and mGlu5). This subgroup of receptors is widely and differentially expressed in neuronal and glial cells within the brain, and receptor activation has been implicated in the control of an array of key signalling events, including roles in the adaptative changes needed for long-term depression or potentiation of neuronal synaptic connectivity. In addition to playing critical physiological roles within the brain, the mGlu receptors are also currently the focus of considerable attention because of their potential as drug targets for the treatment of a variety of neurological and psychiatric disorders.

Animals↗
PubMed2005

Compartmentation of G-protein-coupled receptors and their signalling components in lipid rafts and caveolae.

G-protein-coupled receptors (GPCRs) and post-GPCR signalling components are expressed at low overall abundance in plasma membranes, yet they evoke rapid, high-fidelity responses. Considerable evidence suggests that GPCR signalling components are organized together in membrane microdomains, in particular lipid rafts, enriched in cholesterol and sphingolipids, and caveolae, a subset of lipid rafts that also possess the protein caveolin, whose scaffolding domain may serve as an anchor for signalling components. Caveolae were originally identified based on their morphological appearance but their role in compartmentation of GPCR signalling has been primarily studied by biochemical techniques, such as subcellular fractionation and immunoprecipitation. Our recent studies obtained using both microscopic and biochemical methods with adult cardiac myocytes show expression of caveolin not only in surface sarcolemmal domains but also at, or close to, internal regions located at transverse tubules/sarcoplasmic reticulum. Other results show co-localization in lipid rafts/caveolae of AC (adenylyl cyclase), in particular AC6, certain GPCRs, G-proteins and eNOS (endothelial nitric oxide synthase; NOS3), which generates NO, a modulator of AC6. Existence of multiple caveolin-rich microdomains and their expression of multiple modulators of signalling strengthen the evidence that caveolins and lipid rafts/caveolae organize and regulate GPCR signal transduction in eukaryotic cells.

Animals↗
PubMed2001

Constitutive arrestin-mediated desensitization of a human vasopressin receptor mutant associated with nephrogenic diabetes insipidus.

Agonist-dependent desensitization and internalization of G protein-coupled receptors (GPCR) are mediated by the binding of arrestins to phosphorylated receptors. The affinity of arrestins for the phosphorylated GPCR regulates the ability of the internalized receptor to be dephosphorylated and recycled back to the plasma membrane. In this study, we show that the naturally occurring loss of function vasopressin receptor mutation R137H, which is associated with familial nephrogenic diabetes insipidus, induces constitutive arrestin-mediated desensitization. In contrast to the wild-type vasopressin receptor, the nonsignaling R137H receptor is phosphorylated and sequestered in arrestin-associated intracellular vesicles even in the absence of agonist. Eliminating molecular determinants on the receptor that promote high affinity arrestin-receptor interaction reestablishes plasma membrane localization and the ability of the mutated receptors to signal. These findings suggest that unregulated desensitization can contribute to the etiology of a GPCR-based disease, implying that pharmacological targeting of GPCR desensitization may be therapeutically beneficial.

Adenylyl Cyclases↗
PubMed1996

Dynamin and beta-arrestin reveal distinct mechanisms for G protein-coupled receptor internalization.

The process of agonist-promoted internalization (sequestration) of G protein-coupled receptors (GPCRs) is intimately linked to the regulation of GPCR responsiveness. Following agonist-mediated desensitization, sequestration of GPCR is presumably associated with the dephosphorylation and recycling of functional receptors. However, the exact mechanisms responsible for GPCR sequestration, even for the prototypic beta2-adrenergic receptor (beta2AR), have remained controversial. We demonstrate here that dynamin, a GTPase that regulates the formation and internalization of clathrin-coated vesicles, is essential for the agonist-promoted sequestration of the beta2AR, suggesting that the beta2AR internalizes via the clathrin-coated vesicle-mediated endocytic pathway. In contrast, internalization of the angiotensin II type 1A receptor (AT1AR), another typical GPCR, does not require dynamin. In addition, the AT1AR internalizes independent of the function of beta-arrestin, a critical component for beta2AR cellular trafficking, but additional AT1ARs are mobilized to the dynamin-dependent pathway upon overexpression of beta-arrestin. These findings demonstrate that GPCRs can utilize distinct endocytic pathways, distinguishable by dynamin and beta-arrestin, and that beta-arrestins function as adaptor proteins specifically targeting GPCRs for dynamin-dependent endocytosis via clathrin-coated vesicles.

Animals↗
PubMed1997

A central role for beta-arrestins and clathrin-coated vesicle-mediated endocytosis in beta2-adrenergic receptor resensitization. Differential regulation of receptor resensitization in two distinct cell types.

G protein-coupled receptor (GPCR) sequestration to endosomes is proposed to be the mechanism by which G protein-coupled receptor kinase (GRK)-phosphorylated receptors are dephosphorylated and resensitized. The identification of beta-arrestins as GPCR trafficking molecules suggested that beta-arrestins might represent critical determinants for GPCR resensitization. Therefore, we tested whether beta2-adrenergic receptor (beta2AR) resensitization was dependent upon beta-arrestins and an intact clathrin-coated vesicle endocytic pathway. The overexpression of either the beta-arrestin 1-V53D dominant negative inhibitor of beta2AR sequestration or dynamin I-K44A to block clathrin-coated vesicle-mediated endocytosis impaired both beta2AR dephosphorylation and resensitization. In contrast, resensitization of a sequestration-impaired beta2AR mutant (Y326A) was reestablished following the overexpression of either GRK2 or beta-arrestin 1. Moreover, beta2ARs did not resensitize in COS-7 cells as the consequence of impaired sequestration and dephosphorylation. However, beta2AR resensitization was restored in these cells following the overexpression of beta-arrestin 2. These findings demonstrate, using both loss and gain of function paradigms, that beta2AR dephosphorylation and resensitization are dependent upon an intact sequestration pathway. These studies also indicate that beta-arrestins play an integral role in regulating not only the desensitization and intracellular trafficking of GPCRs but their ability to resensitize. beta-Arrestin expression levels appear to underlie cell type-specific differences in the regulation of GPCR resensitization.

Adenylyl Cyclases↗
PubMed1999

Agonist-dependent phosphorylation of the G protein-coupled receptor kinase 2 (GRK2) by Src tyrosine kinase.

GRK2 is a member of the G protein-coupled receptor kinase (GRK) family, which phosphorylates the activated form of a variety of G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. It has been recently reported that stimulation of the mitogen-activated protein kinase cascade by GPCRs involves tyrosine phosphorylation of docking proteins mediated by members of the Src tyrosine kinase family. In this report, we have investigated the possible role of c-Src in modulating GRK2 function. We demonstrate that c-Src can directly phosphorylate GRK2 on tyrosine residues, as shown by in vitro experiments with purified proteins. The phosphorylation reaction exhibits an apparent K(m) for GRK2 of 12 nM, thus suggesting a physiological relevance in living cells. Consistently, overexpression of the constitutively active c-Src Y527F mutant in COS-7 cells leads to tyrosine phosphorylation of co-expressed GRK2. In addition, GRK2 can be detected in phosphotyrosine immunoprecipitates from HEK-293 cells transiently transfected with this Src mutant. Interestingly, phosphotyrosine immunoblots reveal a rapid and transient increase in GRK2 phosphorylation upon agonist stimulation of beta(2)-adrenergic receptors co-transfected with GRK2 and wild type c-Src in COS-7 cells. This tyrosine phosphorylation is maximal within 5 min of isoproterenol stimulation and reaches values of approximately 5-fold over basal conditions. Furthermore, GRK2 phosphorylation on tyrosine residues promotes an increased kinase activity toward its substrates. Our results suggest that GRK2 phosphorylation by c-Src is inherent to GPCR activation and put forward a new mechanism for the regulation of GPCR signaling.

Adrenergic beta-Agonists↗
PubMed2000

Phosphorylation and regulation of a Gq/11-coupled receptor by casein kinase 1alpha.

Agonist-mediated receptor phosphorylation by one or more of the members of the G-protein receptor kinase (GRK) family is an established model for G-protein-coupled receptor (GPCR) phosphorylation resulting in receptor desensitization. Our recent studies have, however, suggested that an alternative route to GPCR phosphorylation may be an operation involving casein kinase 1alpha (CK1alpha). In the current study we investigate the involvement of CK1alpha in the phosphorylation of the human m3-muscarinic receptor in intact cells. We show that expression of a catalytically inactive mutant of CK1alpha, designed to act in a dominant negative manner, inhibits agonist-mediated receptor phosphorylation by approximately 40% in COS-7 and HEK-293 cells. Furthermore, we present evidence that a peptide corresponding to the third intracellular loop of the m3-muscarinic receptor (Ser(345)-Leu(463)) is an inhibitor of CK1alpha due to its ability to both act as a pseudo-substrate for CK1alpha and form a high affinity complex with CK1alpha. Expression of this peptide was able to reduce both basal and agonist-mediated m3-muscarinic receptor phosphorylation in intact cells. These results support the notion that CK1alpha is able to mediate GPCR phosphorylation in an agonist-dependent manner and that this may provide a novel mechanism for GPCR phosphorylation. The functional role of phosphorylation was investigated using a mutant of the m3-muscarinic receptor that showed an approximately 80% reduction in agonist-mediated phosphorylation. Surprisingly, this mutant underwent agonist-mediated desensitization suggesting that, unlike many GPCRs, desensitization of the m3-muscarinic receptor is not mediated by receptor phosphorylation. The inositol (1,4, 5)-trisphosphate response did, however, appear to be dramatically potentiated in the phosphorylation-deficient mutant indicating that phosphorylation may instead control the magnitude of the initial inositol phosphate response.

Animals↗
PubMed2000

Constitutive signaling by Kaposi's sarcoma-associated herpesvirus G-protein-coupled receptor desensitizes calcium mobilization by other receptors.

We coexpressed Kaposi's sarcoma-associated herpesvirus G protein-coupled receptors (KSHV-GPCRs) with thyrotropin-releasing hormone (TRH) receptors or m1-muscarinic-cholinergic receptors in Xenopus oocytes and in mammalian cells. In oocytes, KSHV-GPCR expression resulted in pronounced (81%) inhibition (heterologous desensitization) of Ca(2+)-activated chloride current responses to TRH and acetylcholine. Similar inhibitions of cytoplasmic free Ca(2+) responses to TRH were observed in human embryonic kidney HEK 293 EM cells and in mouse pituitary AtT20 cells. Further study of oocytes showed that this inhibition was partially reversed by interferon-gamma-inducible protein 10 (IP-10), an inverse agonist of KSHV-GPCR. The basal rate of (45)Ca(2+) efflux in oocytes expressing KSHV-GPCRs was 4.4 times greater than in control oocytes, and IP-10 rapidly inhibited increased (45)Ca(2+) efflux. In the absence of IP-10, growth-related oncogene alpha caused a further 2-fold increase in (45)Ca(2+) efflux. In KSHV-GPCR-expressing oocytes, responses to microinjected inositol 1,4,5-trisphosphate were inhibited by 74%, and this effect was partially reversed by interferon-gamma-inducible protein 10. Treatment with thapsigargin suggested that the pool of calcium available for mobilization by TRH was decreased in oocytes coexpressing KSHV-GPCRs. These results suggest that constitutive signaling by KSHV-GPCR causes heterologous desensitization of responses mediated by other receptors, which signal via the phosphoinositide/calcium pathway, which is caused by depletion of intracellular calcium pools.

Acetylcholine↗
PubMed2001

Rapid protein kinase D translocation in response to G protein-coupled receptor activation. Dependence on protein kinase C.

Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation.

3T3 Cells↗
PubMed2001

Real time analysis of the affinity regulation of alpha 4-integrin. The physiologically activated receptor is intermediate in affinity between resting and Mn(2+) or antibody activation.

This work examines the affinity of alpha(4)beta(1)-integrin and whether affinity regulation by G protein-coupled receptor (GPCR) and chemokines receptors is compatible with cell adhesion mediated between alpha(4)-integrin and vascular cell adhesion molecule-1. We used flow cytometry to examine the binding of a fluorescent derivative of an LDV peptide (Chen, L. L., Whitty, A., Lobb, R. R., Adams, S. P., and Pepinsky, R. B. (1999) J. Biol. Chem. 274, 13167-13175) to several cell lines and leukocytes with alpha(4)-integrin ranging from about 2,000 to 100,000 sites/cell. The results support the idea that alpha(4)-integrins exhibit multiple affinities and that affinity changes are regulated by the dissociation rate and conformation. The affinity varies by 3 orders of magnitude with the affinity induced by binding mAb TS2/16 plus Mn(2+) > Mn(2+) ' TS2/16 > activation because of occupancy of GPCR or chemokines receptor > resting receptors. A significant fraction of the receptors respond to the activating process. The change in alpha(4)-integrin affinity and the corresponding change in off rates mediated by GPCR receptor activation are rapid and transient, and their duration depends on GPCR desensitization. The affinity changes mediated by IgE receptor or interleukin-5 receptor persist longer. It appears that the physiologically active state of the alpha(4)-integrin, determined by inside-out signaling, has similar affinity in several cell types.

Antibodies, Monoclonal↗
PubMed2001

Regulated nucleocytoplasmic transport of protein kinase D in response to G protein-coupled receptor activation.

Protein kinase D (PKD)/protein kinase C mu is a serine/threonine protein kinase activated by growth factors, antigen-receptor engagement, and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires protein kinase C (PKC) activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular distribution of PKD was analyzed in live cells by imaging fluorescent protein-tagged PKD and in fixed cells by immunocytochemistry. We found that PKD shuttled between the cytoplasm and the nucleus in both fibroblasts and epithelial cells. Cell stimulation with mitogenic GPCR agonists that activate PKD induced a transient nuclear accumulation of PKD that was prevented by inhibiting PKC activity. The nuclear import of PKD requires its cys2 domain in conjunction with a nuclear import receptor, while its nuclear export requires its pleckstrin homology domain and a competent Crm1-dependent nuclear export pathway. This study thus characterizes the regulated nuclear transport of a signaling molecule in response to mitogenic GPCR agonists and positions PKD as a serine kinase whose kinase activity and intracellular localization is coordinated by PKC.

3T3 Cells↗
PubMed2002

A novel protein-protein interaction between a G protein-coupled receptor and the phosphatase SHP-2 is involved in bradykinin-induced inhibition of cell proliferation.

Mitogenic G protein-coupled receptor (GPCR) signaling has been extensively studied. In contrast, little is known about anti-mitogenic GPCR signaling. We show here that anti-mitogenic signaling of a GPCR, the bradykinin B2 receptor, involves a novel direct protein-protein interaction. The antiproliferative effect of bradykinin was accompanied by a transient increase in protein-tyrosine phosphatase activity. Using surface plasmon resonance analysis, we observed that an immunoreceptor tyrosine-based inhibitory motif (ITIM) located in the C-terminal part of the B2 receptor interacted specifically with the protein-tyrosine phosphatase SHP-2. The interaction was confirmed in primary culture renal mesangial cells by co-immunoprecipitation of a B2 receptor.SHP-2 complex. The extent of the interaction was transiently increased by stimulation with bradykinin, which was accompanied by an increase in specific SHP-2 phosphatase activity. Mutational analysis of the key ITIM residue confirmed that the B2 receptor ITIM sequence is required for interaction with SHP-2, SHP-2 activation, and the anti-mitogenic effect of bradykinin. Finally, in mesangial cells transfected with a dominant-negative form of SHP-2, bradykinin lost the ability to inhibit cell proliferation. These observations demonstrate that bradykinin inhibits cell proliferation by a novel mechanism involving a direct protein-protein interaction between a GPCR (the B2 receptor) and SHP-2.

Amino Acid Sequence↗
PubMed2003

A G protein-coupled receptor kinase induces Xenopus oocyte maturation.

Several recent studies have suggested that resumption of oocyte meiosis, indicated by germinal vesicle breakdown or GVBD, involves inhibition of endogenous heterotrimeric G proteins in both frogs and mice. These studies imply that a heterotrimeric G protein(s), and hence its upstream activator (a G protein-coupled receptor or GpCR), is activated in prophase oocytes and is responsible for maintaining meiosis arrest. To test the existence and function of this putative GpCR, we utilized a mammalian G-protein-coupled receptor kinase (GRK3) and beta-arrestin-2, which together are known to cause GpCR desensitization. Injection of mRNA for rat GRK3 caused hormone-independent GVBD. The kinase activity of GRK3 was essential for GVBD induction as its kinase-dead mutant (GRK3-K220R) was completely ineffective. Another GRK3 mutant (GRK3-DeltaC), which lacked the C-terminal G(betagamma)-binding domain and which was not associated with oocyte membranes, also failed to induce GVBD. Furthermore, injection of rat beta-arrestin-2 mRNA also induced hormone-independent GVBD. Several inhibitors of clathrin-mediated receptor endocytosis (the clathrin-binding domain of beta-arrestin-2, concanavalin A, and monodansyl cadaverine) significantly reduced the abilities of GRK3/beta-arrestin-2 to induce GVBD. These results support the central role of a yet-unidentified GpCR in maintaining prophase arrest in frog oocytes and provide a potential means for its molecular identification.

Animals↗
PubMed2003

Promiscuous coupling at receptor-Galpha fusion proteins. The receptor of one covalent complex interacts with the alpha-subunit of another.

Fusion proteins between heptahelical receptors (GPCR) and G protein alpha-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 beta(2) adrenergic and dopamine 1 receptors with G(sL)) and some mutated analogs carrying different tags and defective GPCR or Galpha subunits. Using cotransfection experiments with readout protocols able to distinguish activation at fused and non-fused alpha-subunits, we found that both the GPCR and the Galpha 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-Galpha 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 alpha-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 Galpha to the membrane.

Animals↗
PubMed2003

MAPK-dependent degradation of G protein-coupled receptor kinase 2.

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). Altered expression of GRK2 has been described to occur during pathological conditions characterized by impaired GPCR signaling. We have reported recently that GRK2 is rapidly degraded by the proteasome pathway and that beta-arrestin function and Src-mediated phosphorylation are involved in targeting GRK2 for proteolysis. In this report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2 turnover by the proteasome pathway. Modulation of MAPK activation alters the degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated modulation of wild-type GRK2 stability requires beta-arrestin function and is facilitated by previous phosphorylation of GRK2 on tyrosine residues by c-Src. Consistent with an important physiological role, interfering with this GRK2 degradation process results in altered GPCR responsiveness. Our data suggest that both c-Src and MAPK-mediated phosphorylation would contribute to modulate GRK2 degradation, and put forward the existence of new feedback mechanisms connecting MAPK cascades and GPCR signaling.

Animals↗
PubMed2003

Sequential binding of agonists to the beta2 adrenoceptor. Kinetic evidence for intermediate conformational states.

The beta2 adrenoreceptor (beta2AR) is a prototypical G protein-coupled receptor (GPCR) activated by catecholamines. Agonist activation of GPCRs leads to sequential interactions with heterotrimeric G proteins, which activate cellular signaling cascades, and with GPCR kinases and arrestins, which attenuate GPCR-mediated signaling. We used fluorescence spectroscopy to monitor catecholamine-induced conformational changes in purified beta2AR. Here we show that upon catecholamine binding, beta2ARs undergo transitions to two kinetically distinguishable conformational states. Using a panel of chemically related catechol derivatives, we identified the specific chemical groups on the agonist responsible for the rapid and slow conformational changes in the receptor. The conformational changes observed in our biophysical assay were correlated with biologic responses in cellular assays. Dopamine, which induces only a rapid conformational change, is efficient at activating Gs but not receptor internalization. In contrast, norepinephrine and epinephrine, which induce both rapid and slow conformational changes, are efficient at activating Gs and receptor internalization. These results support a mechanistic model for GPCR activation where contacts between the receptor and structural determinants of the agonist stabilize a succession of conformational states with distinct cellular functions.

Adrenergic beta-Agonists↗