G-protein activation rate

With focus on Gq activation by histamine bound H1R

A study has revealed association kinetics of the G_αq and G_γ2 subunits through H1 histamine receptor (H1R) activation in live HeLa cells by FRET method with engineered monomeric Turquoise (mTqΔ6)-Gαq and YFP-Gγ2 (Adjobo-Hermans MJW et al. 2011). The values obtained are apparently comparative to previously determined figures for Gαs and Gαi by other groups; however, the differences in cell types (HeLa in the study and HEK293 in the other two studies) might not allow parallel comparisons as appropriate if the cells were to differ in factors influencing the associations in detail.

Summarised below are G-protein activation kinetics for the three Gα subunits: Gs, Gi and Gq, determined in the three experiments.

Time constants of G-protein activation:

Gq (Adjobo-Hermans MJW et al. 2011)

In the HeLa cells over-expressing H1R, activation rate of Gq was measured as G-protein subunits association in response to histamine. A laser-scanning microscope with a resonant scanner was employed to perform the ratio imaging of FRET (a frame rate .064 second: about 16 frames per second). By monoexponential curve fitting of the average ratio change, half-time for the activation was determined to be ≈ 350 ms.

The GDP dissociation rate was reported as rate-limiting (1.5 per second), in contrast to an in vitro study, which found GTP exchange to be rate-limiting (Mukhopadhyay & Ross 1999).

Markedly different kinetics were reported for HeLa cells expressing H1R at endogenous level (figures not given).

Gs (Hein et al. 2006)

The activation half-time determined were roughly 450 ms. The measurement was taken with Gs-YFP and CFP-Gγ through A2AR activation by adenosine (100 μM) and β1-adrenoceptor activated by noradrenaline (100 μM) in HEK293T cells. The rates determined were 493 ± 31 ms and 437 ± 54 ms, respectively.

The study also quantified receptor-G protein interaction, with YFP conjugated to each receptor C-terminally and CFP-Gγ. The saturating [agonist] (1mM) was chosen to ensure that diffusion of the agonist did not become time-limiting. A time constant obtained were 49.8 ± 5.5 ms for A2AR, 58.1 ± 7.5 ms for β1-adrenoceptor. After removal of agonist, the dissociation kinetics determined was 14.8 ± 1.6 s for A2AR, and 8.4 ± 1.0 s for β1-adrenoceptor.

Gi (Bunemann, Frank & Lohse 2003)

The group studied Gi activation kinetics via α2A-adrenoceptor in response to noradrenaline (1 μM) stimulation in HEK293 cells. They introduced enhanced (F46L)YFP at position 92 of Ptx-insensitive rat (C351I)Gi1, also constructed two fused CFPs, each to the N-terminus of Gβ or to the C-terminus of Gγ (both human). Based on experimental observations on behaviours of differently fused subunits upon co-transfection, the authors suggested that in intact HEK293 cells the rearrangement of G-protein subunits likely occur during activation prior to dissociation of the subunits. After agonist withdrawal, the FRET signal determined for Gi1-YFP and CFP-Gβ decreased with a half-life of ≈ 38 s. The activation and deactivation time determined for Gi1-YFP and Gγ-CFP was 0.69 ± .03 s and 29.7 ± 2 s, respectively.

The relatively lower time constant obtained for Gi in the study above might be due to the agonist diffusion, for relatively lower [agonist] was applied in the study compared to the other studies.

The Gq activation by histamine-bound H1R:

Adjobo-Hermans MJW, Goedhart J, van Weeren L, Nijmeijer S, Manders EMM, Offermanns S, Gadella Jr WJ. 2011. Real-time visualisation of heteromeric G protein Gq activation in living cells. BMC Biology 9:32.

Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands

The Gq Sensor Construction

They made a sensitive sensor with two chimeric proteins: Gq containing mTq with 6 missing residues at its C-tail (mTqΔ6) inserted into the αB-αC loop; and a yellow fluorescent protein (YFP) conjugated at the N-terminus of Gγ2. The functionality of the sensor was tested in a mouse embryonic fibroblast (MEF) cell-line and a MEF cell-line derived from Gαq/11 deficient mice (MEFq/11-/-). The agonist-stimulation of Gq-coupled bradykinin receptor (BK2), expressed endogenously on the cells, showed elevated [calcium], confirming that the chimeras were functional. The association rate was not investigated in this cell.

The Gq sensor in HeLa cells

Receptor density: H1R was over-expressed in HeLa cells (radioligand binding sites were 710 fmol per mg; basal level at 150 fmol per mg for mock-transfected). The expression of mTqΔ6-Gq was confirmed by immunoblotting to be at an equivalent level to the endogenous Gq.

Gβ1 for the sensor: When EYFP-Gβ1 was employed as acceptor instead of YFP-Gγ2, the FRET change detected was much less.

Ligand concentrations: The agonist histamine (100 μM) induced the changes in intensity with about twice larger amplitude; increased intensity of the donor mTq and decreased emission of the acceptor indicated changes in orientation of the Gq αB-αC loop and the N-terminus of Gγ2. With H1R over-expression along with a prolonged histamine treatment, the activation of Gq remained for at least 8 min. The sensor was deactivated by the addition of an inverse agonist, mepyramine (10 μM). A transient FRET change was seen when stimulated with 0.1 μM histamine; further increases in [histamine] sustained its duration (Figure 3 in the publication).

The authors also noted that the prolonged histamine treatment caused active Gq to accumulate progressively, and the desensitisation of active Gq becomes less efficient by each increase in [histamine].

The effect of RGS: To clarify why the activation state of Gq was prolonged in H1R over-expressed HeLa cells, the group assessed the effect of RGS proteins in desensitisation of Gq by making mTqΔ6-(G188S)Gq, insensitive to RGS. The slower onset of Gq activation and reduced desensitisation was observed.

The effect of p63RhoGEF: Co-expression of p63RhoGEF increased the ratio changes in response to histamine (Figure 5C in the paper); the effect was reversed by mepyramine. The positive effect was not observed for a mutant (L475A)p63RhoGEF which doesn’t bind to Gq.

Quantitative FLIM technique: To determine the excited state life-time of the donor fluorophores, frequency-domain FLIM was employed to measure two lifetimes, namely the phase lifetime and the modulation lifetime:

the modulation lifetime of mTq: reduced (values not given); the phase lifetime: 3.4 ns for donor and 2.5 ns with acceptor. FRET efficiency was estimated to be 26%. With H1R, the efficiencies were similar at ground state, but the donor lifetime increased when H1R was activated by histamine, and also by p63RhoGEF co-expressed.

Ligand-independent, mechanosensitive activation of H1R: Mechanosensitive activation of H1R has previously been reported (Medros y Schnitzler et al. 2008). The group applied hypotonic stimulation to HeLa cells in order to measure the Gq activation. The effect was insignificant for endogenous H1R. In H1R over-expressed cells, hypotonic stimulus caused Gq activation (Figure 7 in the paper), and PH domain of PLCδ1 was translocated to the cytosol; the effect was unaffected by mepyramine.

References

Adjobo-Hermans MJW, Goedhart J, van Weeren L, Nijmeijer S, Manders EMM, Offermanns S, Gadella Jr WJ. 2011. Real-time visualisation of heteromeric G protein Gq activation in living cells. BMC Biology 9:32.

Bunemann M, Frank M, Lohse MJ. 2003. Gi protein activation in intact cells involves subunit rearrangement rather than dissociation. PNAS USA 100(26):16077-16082.

Hein P, Rochais F, Hoffmann C, Dorsch S, Nikolaev VO, Engelhardt S, Berlot CH, Lohse MJ, Bunemann M. 2006. Gs activation is time-limiting in initiating receptor-mediated signaling. J Biol Chem 281(44):33345-33351.

Mederos y Schnitzler M, Storch U, Meibers S, Nurwakagari P, Breit A, Essin K, Gollasch M, Gudermann T. 2008. Gq-coupled receptors as mechanosensors mediating myogenic vasoconstriction. EMBO J. 27(23):3092-3103.

Mukhopadhyay S, Ross EM. 1999. Rapid GTP binding and hydrolysis by G(q) promoted by receptor and GTPase-activating proteins. PNAS USA. 96(17):9539-9544.

Crystal Structure of the Active State Complex with Nucleotide-Free G-Protein Trimer

It is truly an astonishing moment seeing something dying to see it for sometime, as well as witnessing a magnificent achievement. For a first time, the crystal structure of G-protein coupled receptor ternary complex, obtained in a combined efforts between the groups in the USA, Denmark and Ireland, has been revealed - making another milestone which has finally been fixed in the history of receptor pharmacology.

The study published in Nature is:

Crystal structure of the β2 adrenergic receptor-Gs protein complex
Søren G. F. Rasmussen 1,2, Brian T. DeVree 3, Yaozhong Zou 1, Andrew C. Kruse 1, Ka Young Chung 1, Tong Sun Kobilka 1, Foon Sun Thian 1, Pil Seok Chae 4, Els Pardon 5,6, Diane Calinski 3, Jesper M. Mathiesen 1, Syed T. A. Shah 7, Joseph A. Lyons 7, Martin Caffrey 7, Samuel H. Gellman 4, Jan Steyaert 5,6, Georgios Skiniotis 8, William I. Weis 1,9, Roger K. Sunahara 3 & Brian K. Kobilka 1.
1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA. 2. Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark. 3. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. 4. Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA. 5. Department of Molecular and Cellular Interactions, Vlaams Instituut voor Biotechnologie (VIB), Vrije Universiteit Brussel, B-1050 Brussel, Belgium. 6. Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium. 7. Membrane Structural and Functional Biology Group, Schools of Medicine and Biochemistry & Immunology, Trinity College, Dublin 2, Ireland. 8. Life Sciences Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA. 9. Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA.

Many congratulations to the groups above for their successes in obtaining this, and thank you all the above for making it possible to see it.

Brief Summary

PDB file: 3SN6

Method: x-ray diffraction

Resolution: 3.20 angstrom

The complex comprising five polymers:
1. Bovine Gαs short isoform (380 amino acids)
2. Rat Gβ1 GI/GS/GT (351 a.a.)
3. Bovine Gγ2 GI/GS/GT (68 a.a.)
4. Enterobacteriophage T4 Lysozme--Human Beta-2 Adrenoceptor chimera (514 a.a.)
5. Llama Camelid antibody VHH fragment (138 a.a.) i.e. nanobody Nb35 Gs-binding nanobody Nb35 stabilised the β2AR-Gs complex.

Ligand: the receptor has an agonist BI-167107 bound.

Their journal article published in Nature beautifully illustrates the outward movement of TMs, notably of TM6 (14 angstrom) and to a less degree of TM5 in overlaid crystal structures of the newly obtained active β2AR and the previously obtained inactive counterpart (figure 3 in the original article).

The article reveals the interactions between the receptor and Gs in substantial detail. TM3, TM5 and ICL2 were shown to participate mainly (figure 4 in the article).

They have also compared conformational changes in Gs regarding GTPγS binding, revealing a rotated displacement (6 angstrom) of α5 helix of Gs from nearby ICL2 towards the core of the 7TMs upon GTPγS release (figure 5 in the article).

The article insightfully closes with a proposed sequence of the receptor-Gs complex formation (figure 6 in the article).

Delightedly, there are more information technically relevant in detail to come in following two publications (Westfield et al.; Chung et al. submitted).

Here are some pictures:

The pentameric complex containing T4 lysozyme-human β2AR chimera (yellow), bovine Gs (blue), rat Gβ (cyan), bovine Gγ2 (green) and llama nanobody 35 (red).

The side view facing to TM3-ICL2 and Gs.

















The side view facing TM2,

Gβ (cyan), and Gγ2 (green).

The bound agonist BI-167197 is shown in purple.




















The side view facing TM5.

The side view facing TM7-helix8.





















An extracellular view.
The residues interacting with the agonist are W3.28, N6.55, Y7.35 and N7.39 in Ballesteros-Weinstein numbering scheme.

A tilted view of the above, facing TM5 and TM6. The residues interacting with the agonist are as above and V3.36.

Side view facing TM3. The residues interacting with the agonist are as above, and D3.32, S 5.42 and S5.46.

The side view facing TM7. The agonist interacting with the aforementioned residues and F6.52.

Cytosolic view.

A distinctive activation mechanism of a Family B orphan GPR56 by relocating its shed N-terminal domain

A study on GPR56, belonging to a subfamily of an adhesion GPCR (a.k.a. LN-7TM) of Family B, found that this receptor can be activated by removing its large N-terminal domain. The N-terminal domain (NT) is proteolytically cleaved in a process termed receptor shedding. The cleaved NT associates with the receptor as it maintains itself at an inactive state.
In their suggested model of GPR56 activation, the receptor generates signals upon NT dissociation by means of either: heteromeric interaction between NT and its ligand unidentified; and/or homophilic associations between local NTs.

The GPR56 N-Terminus Controls Receptor Signaling Activity 2011. JBC in press.
KJ Paavola, JR Stephenson, SL Ritter, SP Alter & RA Hall
Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, USA

The study demonstrated:
1. The shed NT of GPR56 (GPR56-NT) associates with the receptor.

2. The NT truncation of GPR56 enhances receptor induction of Rho signalling. The truncated receptors (ΔN-342) showed notably enhanced β-arrestin binding, and were subjected to extensive ubiquitination. The increased cytotoxicity (measured by LDH levels) by the truncated was observed but was reversed by an increase in β-arrestin2.

3. GPR56-NT can homomerise.

Relevance to other studies:
The findings of this study provides an explanatory evidence for an observation made in other study: anti-GPR56 polyclonal antibodies raised against the N-terminal domain of GPR56 activated Rho pathway (Iguchi et al. 2008). Homodimerisation of N-terminal domains have also been documented for other member of the LN-7TM subfamily: Cadherin EGF LAG seven-pass G-type receptor (Celsr) type 2 or type 3 involved in neurite growth (Shima et al. 2007); the N-terminal domain of Celsr2/3 are enormous, over 2000 and over 3000 amino acid lengths respectively.

About GPR56:

Human GPR56 on uniprot
Its N-terminal region comprises 377 amino acids following a signalling peptide of 25 amino acid lengths. Numerous Ser/Thr residues (≈16% of the 377 residues) could be glycosylated in the Golgi apparatus. The N-terminal region contains a GPCR proteolytic site (GPS) located by TM1.

Mutations in GPR56 and the Roles of GPR56 in CNS
Missense mutations in GPS (C346S and W349S) could impair the proteolytic cleavage, and the mutant receptors were found not to traffic beyond the endoplasmic reticulum (Jin et al. 2007).

Loss-of-function mutations in GPR65 result in bilateral frontoparietal polymicrogyria (BFPP) (Piao et al. 2004), an autosomal recessive disorder affecting brain development (see a note below). All identified BFPP-related mutations are in the extracellular domains of the receptor, affecting its cell-surface expression and/or receptor shedding (Jin et al. 2007; Chlang et al. 2011).

Bilateral frontoparietal polymicrogyria (BFPP)
A congenital brain malformation displaying abnormal groove patterning on the surface of the cortex with numerous but smaller gyri. It causes developmental delay in both mental and motor skills, and individuals affected may suffer from epilepsy.
For more information, see a review by Jansen & Andermann 2005.

GPR56 also regulates granule cell adhesion during rostral cerebellar development (Koirala et al. 2009).

GPR56 Signalling and Its Regulation
During the development of the forebrain, it regulates neural progenitor cell migration through activation of G12/13 and Rho pathway involving serum-responsive element and NFκβ-responsive element; it also induces F-actin accumulation in NIH3T3 cell-line (Iguchi et al. 2008).

GPR56 has also been shown to interact with Gq/11 through specific tetraspanins, CD9 or CD81 which probably function as scaffolding proteins (Little, Helmer & Stipp, 2004). The study also showed that GPR65 can be internalised in response to phorbol ester (PMA).

GPR56 in Carcinogenesis and Metastasis
GPR56 have been found to over-express in various tumour tissues, involved in cell transformation through cell adhesion pathway (Ke et al. 2007).

However, in metastatic melanoma cells, GPR56 was found to express at low level; moreover, GPR56 exhibited an anti-metastatic effect by interacting with a cross-linking enzyme in the extracellular matrix, known as tissue transglutaminase (TG2), which binds to fibronectin and integrins to facilitate cell-adhesion (Xu et al. 2006; Xu & Hynes 2007).

Remarks:
For having a large impact on cell motility as well as with the demonstrated oncogenic potential, it makes sense for GPR56 to naturally possess an inbuilt antagonistic lid as a preventive measure for non-specific activity induced by any non-specific ligand. The ligand of GPR56 is yet unknown; suppose by an elimination process if it was possible to confirm that the receptor is indeed ligand-less, this mode of action via homodimerisation of the N-terminal regions would be a sole activation mechanism of this receptor. If so, the receptor function would be affected directly by the receptor density, and by the population of transmembrane or membrane-associated proteins present in the local environment. More detailed characterisations of GPR56 are awaited.

References
Chlang NY et al. J Biol Chem. 286(16):14215-25.
Iguchi T et al. 2008. J Biol Chem. 283(21): 14469–14478.
Jansen A & Andermann E. 2005. J Med Genet. 42:369–378.
Jin Z et al. 2007. Hum Mol Genet. 16(16):1972-85.
Ke N et al. 2007. Mol Cancer Ther. 6: 1840-1850.
Koirala S et al. 2009. J Neurosci. 29(23):7439-49.
Little KD, Helmer ME & Stipp CS. 2004. Mol Biol Cell. 15: 2375-2387.
Paavola KJ et al. 2011. J Biol Chem. June 27 in press.
Shima Y et al. 2007. Nat Neurosci 10(8): 963-969
Xu L et al. 2006. PNAS. 103(24): 9023–9028.
Xu L & Hynes RO 2007. Cell Cycle 6(2): 160‐165.