Human regulator of G-protein signalling 1

Target ID:

RGS1A

Aliases:

1R20BL34IER1IR20

Deposition Date:

Monday 20th June 2005

Families:

G-Protein Regulators

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2BV1

Authors:

J.M.ELKINS,X.YANG,M.SOUNDARARAJAN,G.A.SCHOCH,A.HARONITI,M.SUNDSTROM,A.EDWARDS,C.ARROWSMITH,D.A.DOYLE

Site:

Oxford

ICM Datapack:

ICM Datapack

2BV1

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 that consist of the Gα and Gβγ subunits.
In the inactive state, Gα and Gβγ exist as a complex with the Gα GTPase
in an inactive GDP-bound state.
Upon activation GDP is replaced with GTP which induces Gα and Gβγ complex disassociation.
At this stage, both Gα and Gβγ proteins are active.
Gα 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-Gα protein can recombine with Gβγ,
thus terminating the signal.
RGS1 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.

Stimulation of the immune system results in a complex, coordinated signalling process that prepares the body to respond to an infection and elliminated the infection. A major element of this process is the activation of the B-cells which produce the antibodies that target the pathogen for destruction. Within the B-cells several genes are up-regulated upon activation. RGS1 is one that is immediately activated (Newton et al 1993). The GAP activity of RGS1 is directed against Giα and Gqα G-proteins with the result that the direction of B-cell migration is altered (Moratz et al, 2000). RGS1 even alters the movement of B-cells within lymph nodes (Han et al, 2005). Demonstration that RGS1 is also upregulated by bacterial lipopolysaccharides (Panetta et al, 1999) enhances the suggestion that RGS1 plays a key role in the signalling response of the immune system.

Materials and Methods

RGS1

PDB:2BV1

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:RGS 1A-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal hexahistidine tag
Host:BL-21(DE3)R3

Construct

Prelude:
Sequence:SMSGMKSSKSKDVLSAAEVMQWSQSLEKLLANQTGQNVFGSFLKSEFSEENIEFWLACEDYKKTESDLLPCKAEEIYKAFVHSDAAKQINIDFRTRESTAKKIKAPTPTCFDEAQKVIYTLMEKDSYPRFLKSDIYLNLLNDLQA
Vector:pLIC-SGC

Growth

Medium:
Antibiotics:
Procedure:

Purification

Procedure
Column 1: Low pressure chromatography using Bio-Rad Econo column (2.5 cm x 13 cm).

Buffers: Wash Buffer I (WBI): 50 mM Hepes pH 8.0, 300 mM NaCl, 5 % glycerol, 10 mM imidazole pH 8.0; Wash Buffer II (WBII): 50 mM Tris pH 8.0, 300 mM NaCl, 5 % glycerol, 30 mM imidazole pH 8.0; Elute Buffer (EB): 50 mM Hepes pH 8.0, 300 mM NaCl, 5 % glycerol, 250 mM imidazole pH 8.0.

Procedure: Total volume of Ni-NTA added to BioRad drip column: 4 mL (50 %). Resin washed with 12.5 mL of WBI. The supernatent was applied to a column using 5 mL pipette and allowed to pass over the resin. The flow through was collected in a 50 mL falcon tube and applied once more to the column. Two wash steps followed. Wash with 12.5 mL of WBI. Wash with 12.5 mL column vols of WBII. Elute with 14 mL of EB into 7x2 mL fractions.

Column 2 : Size exclusion using a S75 16/60 column

Buffers : GF Buffer: 50 mM Tris pH 8.0, 500 mM NaCl, 0.5mM TCEP

Procedure: The column was pre-equilibrated with two column volumes of GF buffer (flow rate 1 mL/min). The fractions from gel filtration that contained RGS 1A were pooled and concentrated before loading on to S75 16/60 gel filtration column. The fractions containing protein were identified on a coomasie blue stained gel.

Extraction

Procedure
1 tablet protein inhibitor in 10 mL Lysis Buffer was added to the 1L growth pellet. Total vol: 45 mL (estimate).

Cell breakage: 5 passes through the Emulsiflex C5 high pressure homogeniser. Total vol: 50 mL (estimate).
Concentration:
MassSpec:
Crystallization:Column 1: Low pressure chromatography using Bio-Rad Econo column (2.5 cm x 13 cm).

Column 2 : Size exclusion using a S75 16/60 column

Buffers : GF Buffer: 50 mM Tris pH 8.0, 500 mM NaCl, 0.5mM TCEP

References

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.

Han, S. B., Moratz, C., Huang, N. N., Kelsall, B., Cho, H., Sh,i C. S., Schwartz, O., Kehrl, J. H. (2005). Rgs1 and Gnai2 regulate the entrance of B lymphocytes into lymph nodes and B cell motility within lymph node follicles. Immunity. 22: 343-354.

Moratz, C., Kang, V. H., Druey, K. M., Shi, C. S., Scheschonka, A., Murphy, P. M., Kozasa, T., Kehrl, J. H. (2000). Regulator of G protein signaling 1 (RGS1) markedly impairs Gi alpha signaling responses of B lymphocytes. J Immunol. 164: 1829-1838.

Newton, J. S., Deed, R. W., Mitchell ,E. L., Murphy, J. J., Norton, J. D. (1993). A B cell specific immediate early human gene is located on chromosome band 1q31 and encodes an alpha helical basic phosphoprotein.
Biochim Biophys Acta. 1216: 314-316.

Panetta, R., Guo, Y., Magder, S., Greenwood, M. T. (1999). Regulators of G-protein signaling (RGS) 1 and 16 are induced in response to bacterial lipopolysaccharide and stimulate c-fos promoter expression.
Biochem. Biophys. Res. Commun. 259: 550-556.