scholarly journals G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence

1998 ◽  
Vol 332 (3) ◽  
pp. 781-787 ◽  
Author(s):  
Hisashi KAI ◽  
R. Wayne ALEXANDER ◽  
Masuko USHIO-FUKAI ◽  
P. Reid LYONS ◽  
Marjorie AKERS ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Protein Binding ◽  
G Protein ◽  
G Proteins ◽  
Synthetic Peptides ◽  
Terminal Region ◽  
Functional Coupling ◽  
Intracellular Loop ◽  
Binding Domains ◽  
Gtpase Activation

The vascular angiotensin II type 1 receptor (AT1AR) is a member of the G-protein-coupled receptor superfamily. We mapped the G-protein binding domains of the AT1AR using synthetic peptides selected from the receptor sequence, which interfere with AT1AR–G-protein coupling. Membrane GTPase activity was used as a measure of the functional coupling in rat vascular smooth muscle cells. Peptides corresponding to the N-terminal region of the second intracellular loop (residues 125–137), the N-terminal region of the third intracellular loop (217–227) and the juxtamembranous region of the C-terminal tail (304–316) inhibited angiotensin II-induced GTPase activation by 30%, 30%, and 70%, respectively. The latter two domains (217–227 and 304–316) are predicted to form amphiphilic α-helices. Only the peptide representing residues 217–227 stimulated basal activity (45%). No synthetic peptide had a significant effect on either the number or the affinity of the AT1AR binding. These observations indicate that domains of the second and third regions and the cytoplasmic tail of the AT1AR interact with G-proteins, and that multiple contacts with these receptor domains may be important for binding and activation of the G-proteins.

Site of G protein binding to rhodopsin mapped with synthetic peptides from the alpha subunit

Science ◽  
1988 ◽  
Vol 241 (4867) ◽  
pp. 832-835 ◽  
Author(s):  
H. Hamm ◽  
D Deretic ◽  
A Arendt ◽  
P. Hargrave ◽  
B Koenig ◽  
...  
Keyword(s):  
Protein Binding ◽  
G Protein ◽  
Synthetic Peptides ◽  
Alpha Subunit

scholarly journals Differential reactivity of serum immunoglobulins from Brazilian wild mammals to staphylococcal A and streptococcal G proteins

2012 ◽  
Vol 24 (1) ◽  
pp. 148-152 ◽  
Author(s):  
Afonso Pelli ◽  
Lucio R. Castellano ◽  
Marcos R. S. Cardoso ◽  
Luís A. S. Vasconcelos ◽  
Marcos A. Domingues ◽  
...  
Keyword(s):  
Protein Binding ◽  
G Protein ◽  
G Proteins ◽  
Protein A ◽  
Human Beings ◽  
Human Pathogens ◽  
Wild Mammals ◽  
Detection Systems ◽  
Differential Reactivity ◽  
Human Illness

Human pathogens have evolved to infect vertebrate hosts other than human beings without causing symptoms of the disease, thus permitting them to complete their life cycle and to develop into infectious forms. The identification and management of infected animals are alternatives to control dissemination of the disease and to prevent human illness. In the current study, the potential use of staphylococcal A or streptococcal G proteins was evaluated with enzyme-linked immunosorbent assays (ELISAs) for seroepidemiological studies. Sera were collected from animals that were representative of 23 different Brazilian wild mammals. A high protein A binding rate was observed in all animals, except for the orders Didelphimorphia, Artiodactyla, and Rodentia, in which affinity was medium or low. Affinity for streptococcal G protein was higher in animals of the order Artiodactyla, whereas no streptococcal G protein binding was observed in samples obtained from felines (order Carnivora). Bacterial protein binding to mammalian immunoglobulins was confirmed by immunoblotting. The results suggest that secondary detection systems should be better investigated in ELISA protocols before their implementation in seroepidemiological studies involving wild mammals.

scholarly journals Vasoconstrictor agonists activate G-protein-dependent receptor-operated calcium channels in pig aortic microsomes

1992 ◽  
Vol 282 (1) ◽  
pp. 81-84 ◽  
Author(s):  
L M Blayney ◽  
P W Gapper ◽  
A C Newby
Keyword(s):  
Steady State ◽  
Angiotensin Ii ◽  
Calcium Channels ◽  
G Protein ◽  
G Proteins ◽  
Intact Cells ◽  
Vasoconstrictor Agents ◽  
Receptor Sites ◽  
Alpha Beta ◽  
Ca2 Channels

Receptor-operated Ca2+ channels were characterized by their ability to decrease steady-state ATP-dependent Ca2+ accumulation into pig aortic microsomes. The vasoconstrictor agents noradrenaline, angiotensin II and adenosine 5′-[alpha beta-methylene]triphosphate (pp[CH2]pA) all decreased Ca2+ accumulation only when sonicated into vesicles (to allow access to receptor sites) and in the presence of guanosine 5′-[beta gamma-imido]triphosphate to activate transducing G-proteins. The effect of noradrenaline was inhibited by the alpha 2 antagonist yohimbine, but not by the alpha 1 antagonist prazosin. The effect of none of the agonists was reversed by diltiazem. SK&F 96365 (an inhibitor of receptor-mediated Ca2+ influx into intact cells) reversed the effect of noradrenaline, but not that of pp[CH2]pA, which suggests that at least two receptor-operated channels may be present in this preparation.

scholarly journals A Novel Nuclear Signaling Pathway for Thromboxane A2 Receptors in Oligodendrocytes: Evidence for Signaling Compartmentalization during Differentiation

2008 ◽  
Vol 28 (20) ◽  
pp. 6329-6341 ◽  
Author(s):  
Fozia Mir ◽  
Guy C. Le Breton
Keyword(s):  
Signaling Pathway ◽  
G Protein ◽  
G Proteins ◽  
Protein Complexes ◽  
Functional Coupling ◽  
Protein Signaling ◽  
Creb Phosphorylation ◽  
Expression Levels ◽  
Nuclear Signaling ◽  
Subcellular Compartmentalization

ABSTRACT The present study investigated G protein expression, localization, and functional coupling to thromboxane A2 receptors (TPRs) during oligodendrocyte (OLG) development. It was found that as OLGs mature, the expression levels of Gq increase while those of G13 decrease. In contrast, the expression levels of Gs, Go, and Gi do not change significantly. Localization studies revealed that Gq, G13, and Gi are present only in the extranuclear compartment, whereas Gs and Go are found in both the extranuclear and the nuclear compartments. Purification of TPR-G protein complexes demonstrated that TPRs couple to both Gq and G13 in the extranuclear compartment but only to Gs in the nuclear compartment. Furthermore, functional analysis revealed that stimulation of nuclear TPR in OLGs stimulates CREB phosphorylation and myelin basic protein transcription and increases survival. Collectively, these results demonstrate that (i) OLGs selectively modulate the expression of certain G proteins during development, (ii) G proteins are differentially localized in OLGs leading to subcellular compartmentalization, (iii) TPRs couple to Gq and G13 in the extranuclear compartment and to Gs only in the nucleus, (iv) mature OLGs have a functional nuclear TPR-Gs signaling pathway, and (v) nuclear TPR signaling can stimulate CREB phosphorylation and myelin gene transcription and increase cell survival. These findings represent a novel paradigm for selective modulation of G protein-coupled receptor-G protein signaling during cell development.

Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors

2018 ◽  
Vol 11 (559) ◽  
pp. eaat1631 ◽  
Author(s):  
Yoon Namkung ◽  
Christian LeGouill ◽  
Sahil Kumar ◽  
Yubo Cao ◽  
Larissa B. Teixeira ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
G Protein ◽  
G Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Functional Selectivity ◽  
Gpcr Signaling ◽  
Downstream Signaling ◽  
Protein Levels

G protein–coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and β-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gαq and β-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling.

Polarity of TRH receptors in transfected MDCK cells is independent of endocytosis signals and G protein coupling

1996 ◽  
Vol 270 (3) ◽  
pp. C753-C762 ◽  
Author(s):  
C. Yeaman ◽  
M. Heinflink ◽  
E. Falck-Pedersen ◽  
E. Rodriguez-Boulan ◽  
M. C. Gershengorn
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mdck Cells ◽  
Cell System ◽  
Heterotrimeric G Proteins ◽  
Intracellular Loop ◽  
Surface Receptors ◽  
Model Cell ◽  
G Protein Coupled ◽  
Trh Receptors

Information concerning the molecular sorting of G protein-coupled receptors in polarized epithelial cells is limited. Therefore, we have expressed the receptor for thyrotropin-releasing hormone (TRH) in Madin-Darby canine kidney (MDCK) cells by adenovirus-mediated gene transfer to determine its distribution in a model cell system and to begin analyzing the molecular information responsible for its distribution. Equilibrium binding of [methyl-3H]TRH to apical and basolateral surfaces of polarized MDCK cells reveals that TRH receptors are expressed predominantly (>80%) on the basolateral cell surface. Receptors undergo rapid endocytosis following agonist binding; up to 80% are internalized in 15 min. A mutant receptor missing the last 59 residues, C335Stop, is poorly internalized (<10%) but is nevertheless basolaterally expressed (>85%). A second mutant TRH receptor, delta218-263, lacks essentially all of the third intracellular loop and is not coupled to G proteins on binding agonist. This receptor internalizes TRH approximately half as efficiently as wild-type TRH receptors but is nevertheless strongly polarized to the basolateral surface (>90%). These results indicate that molecular sequences responsible for basolateral accumulation of TRH receptors can be segregated from signals for ligand-induced receptor endocytosis and coupling to heterotrimeric G proteins.

scholarly journals Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

2020 ◽  
Author(s):  
Ali I. Kaya ◽  
Nicole A. Perry ◽  
Vsevolod V. Gurevich ◽  
T.M. Iverson
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Dopamine D1 Receptor ◽  
D1 Receptor ◽  
Signaling Cascades ◽  
Intracellular Loop ◽  
Receptor Phosphorylation ◽  
Downstream Signaling ◽  
Fundamental Information ◽  
Src Activation

AbstractAgonist-activated G protein-coupled receptors (GPCRs) must correctly select from hundreds of potential downstream signaling cascades and effectors. To accomplish this, GPCRs first bind to an intermediary signaling protein, such as G protein or arrestin. These intermediaries initiate signaling cascades that promote the activity of different effectors, including several protein kinases. The relative roles of G proteins versus arrestins in initiating and directing signaling is hotly debated, and it remains unclear how the correct final signaling pathway is chosen given the ready availability of protein partners. Here, we begin to deconvolute the process of signal bias from the dopamine D1 receptor (D1R) by exploring factors that promote the activation of ERK1/2 or Src, the kinases that lead to cell growth and proliferation. We found that ERK1/2 activation involves both arrestin and Gαs, while Src activation depends solely on arrestin. Interestingly, we found that the phosphorylation pattern influences both arrestin and Gαs coupling, suggesting an additional way the cells regulate G protein signaling. The phosphorylation sites in the D1R intracellular loop 3 are particularly important for directing the binding of G protein versus arrestin and for selecting between the activation of ERK1/2 and Src. Collectively, these studies correlate functional outcomes with a physical basis for signaling bias and provide fundamental information on how GPCR signaling is directed.Significance StatementThe functional importance of receptor phosphorylation in GPCR regulation has been demonstrated. Over the past decade, the phospho-barcode concept was developed to explain the multi-dimensional nature of the arrestin-dependent signaling network downstream of GPCRs. Here, we used the dopamine-1 receptor (D1R) to explore the effect of receptor phosphorylation on G protein-dependent and arrestin-dependent ERK and Src activation. Our studies suggest that D1R intracellular loop-3 phosphorylation affects both G proteins and arrestins. Differential D1R phosphorylation can direct signaling toward ERK or Src activation. This implies that phosphorylation induces different conformations of receptor and/or bound arrestin to initiate or select different cellular signaling pathways.

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