Human regulator of G-protein signalling 8

Target ID:

RGS8A

Aliases:

MGC119067MGC119068MGC119069

Deposition Date:

Tuesday 26th September 2006

Families:

G-Protein Regulators

Related Diseases:

SignallingNeurobiologyCancer

Structure type:

apo

Download Coordinates:

2IHD

Authors:

A.P.TURNBULL,E.PAPAGRIGORIOU,E.UGOCHUKWU,E.SALAH,C.GILEADI,N.BURGESS,C.BHATIA,O.GILEADI,J.BRAY,J.ELKINS,F.VON DELFT,J.WEIGELT,A.EDWARDS,C.ARROWSMITH,M.SUNDSTROM,D.DOYLE,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2IHD

tabs

Structure Details

Regulators of G-protein signalling (RGS) are domains that terminate the signal emanating from G-protein coupled receptors (GPCR). When a GPCR binds a specific ligand a signal is generated that is transmitted to the G-proteins consisting of the Ga and Gbg subunits.
In the inactive state, Ga and Gbg exist as a complex with the Ga GTPase in an inactive GDP bound state. Upon activation, GDP is replaced with GTP which induces Ga and Gbg complex disassociation. At this stage, both Ga and Gbg proteins are active. Ga does have an intrinsic GTPase activity but this is greatly enhanced by the association with an RGS domain. Hence RGS proteins act as GTPase activating proteins (GAPs). Once GTP hydrolysis has taken place, the GDP-Ga protein can recombine with Gbg thus terminating the signal.

RGS8 is a member of the B/R4 subfamily of RGS proteins. All of these subfamily members have an N-terminal amphipathic helix which enhances the localisation of the protein to the membrane surface (Bernstein et al, 2000; Chen et al, 1999) where it is ideally placed to interact with the G-proteins.

RGS8 modulates the activity of the G-protein coupled inward rectifying K+ channel Kir3.1/Kir3.2 (GIRK1/2; Doupnik et al, 1997; Saitoh et al, 1997). It is the Gbg subunits that bind directly to GIRK, activating the channel (Logothetis et al, 1987). As described above, RGS8 does have a GAP activity through its RGS domain, resulting in deactivation of the GIRK channel however RGS8 also enhances channel activity which has been attributed to its N-terminal region (Jeong & Ikeda, 2001). Explanations for this behaviour have been accounted for through the generation of macromolecular assemblies involving ion channels, G-proteins, GPCR and RGS proteins (Benians et al, 2005; Lavine et al, 2002; Riven et al, 2006).

Materials and Methods

RGS8

PDB:2IHD

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:RGS8A-s001
Entry Clone Source:IMAGE
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)R3 (Phage resistant strain)

Construct

Prelude:
Sequence: mhhhhhhssgvdlgtenlyfqsmLKRLST EEATRWADSFDVLLSHKYGVAAFRAFLKT EFSEENLEFWLACEEFKKTRSTAKLVSKA HRIFEEFVDVQAPREVNIDFQTREATRKN LQEPSLTCFDQAQGKVHSLMEKDSYPRFL RSKMYLDLLS
Vector:Vector: pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Using a glycerol stock of the transformed cells, a starter culture (50 ml of TB plus 50 µg/ml kanamycin) was grown overnight at 37°C (200 rpm). 10 mls of this culture was used to inoculate 3 x 1 litres of Terrific Broth plus 50 µg/ml kanamycin. The cells were further grown at 37°C (200 rpm) until the culture reached an OD600 of between 2-3. At this stage the temperature of the incubator was dropped to 18°C for 1/2 hour before induction of protein synthesis by the addition of 0.2 mM IPTG. Total induction time was ~16 hour after which the cells were pelleted by centrifugation at 6000 rpm for 20 minutes at 4°C. The pellets were then resuspended in stored in a -80°C freezer until further use.

Purification

Procedure
Column 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham)
Procedure: The cell extract was loaded on the column at 0.8 ml/min on an AKTA-express system (GE/Amersham). The column was then washed with 10 column volumes of Binding Buffer, 10 column volumes of Wash Buffer and the RGS8 domain eluted with Elution Buffer at 0.8 ml/min. The eluted peak of A280 was automatically collected.

Column 2 : Gel filtration, Hiload 16/60, S75 16/60 - 120 ml
The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in Gel Filtration Buffer at 1.0 ml/min. Eluted proteins were collected in 1 ml fractions.

Concentration : The eluted fractions that contained the RGS8 domain as judged by SDS-PAGE were pooled and concentrated to 10 mg/ml using a Millipore 10 kD cutoff spin concentrator. The concentrated protein was distributed into 7 x 50 m l aliquots before freezing in a -80°C freezer.

Extraction

Procedure
The 3 litre pellets were resuspended in ~100 ml lysis buffer. The cells were lysed by passing through a high pressure homogeniser ( EmulsiFlex C5), 4 cycles, bringing the final volume to ~150 ml .
PEI (stock 5%) was added to the homogenate to a final concentration of 0.15%. The cell debris, nuclei and DNA were spun down at 16,500 rpm for 60 min (rotor JA 17). The supernatant was collected and filtered through a 0.22 µm filter, using a pump. The last drops that didn't go through were filtered manually through two filters 1.2 plus 0.22µm on a syringe.
Concentration:
Ligand
MassSpec:Expected MWt: 18202.4; Measured MWt: 18203.
Crystallization:Crystals grew from a 1:1 ratio mix of MPDZA-p001-to-reservoir (0.2 M NaI; 20 % PEG 3350 ; 10 % ethylene glycol)
NMR Spectroscopy:
Data Collection:Resolution: 1.7 Å; X-ray source: Synchrotron SLS-X10, single wavelength.
Data Processing:

References

Benians A, Nobles M, Hosny S, Tinker A. (2005). Regulators of G-protein signaling form a quaternary complex with the agonist, receptor, and G-protein. A novel explanation for the acceleration of signaling activation kinetics. J. Biol. Chem. 280: 13383-13394.

Bernstein, L. S., Grillo, A. A., Loranger, S. S., Linder, M. E. (2000). RGS4 binds to membranes through an amphipathic alpha -helix. J. Biol. Chem. 275: 18520-18526.

Chen, C., Seow, K. T., Guo, K., Yaw, L. P., Lin, S. C. (1999). The membrane association domain of RGS16 contains unique amphipathic features that are conserved in RGS4 and RGS5. J. Biol. Chem. 274:19799-19806.

Doupnik CA, Davidson N, Lester HA, Kofuji P. (1997). RGS proteins reconstitute the rapid gating kinetics of gbetagamma-activated inwardly rectifying K+ channels. Proc. Natl. Acad. Sci. U S A. 94: 10461-10466.

Jeong SW, Ikeda SR. (2001).Differential regulation of G protein-gated inwardly rectifying K(+) channel kinetics by distinct domains of RGS8. J. Physiol. 535: 335-347.

Lavine N, Ethier N, Oak JN, Pei L, Liu F, Trieu P, Rebois RV, Bouvier M, Hebert TE, Van Tol HH. (2002). G protein-coupled receptors form stable complexes with inwardly rectifying potassium channels and adenylyl cyclase. J. Biol. Chem. 277: 46010-46019.

Logothetis DE, Kurachi Y, Galper J, Neer EJ, Clapham DE. (1987). The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature. 325: 321-326.

Riven I, Iwanir S, Reuveny E. (2006). GIRK Channel Activation Involves a Local Rearrangement of a Preformed G Protein Channel Complex. Neuron. 51: 561-573.

Saitoh O, Kubo Y, Miyatani Y, Asano T, Nakata H. (1997). RGS8 accelerates G-protein-mediated modulation of K+ currents. Nature. 390: 525-529.