Human regulator of G-protein signalling 16

Target ID:

RGS16A

Aliases:

A28-RGS14A28-RGS14PRGS-R

Deposition Date:

Wednesday 25th May 2005

Families:

G-Protein Regulators

Related Diseases:

SignallingNeurobiology

Structure type:

apo

Download Coordinates:

2BT2

Authors:

G.BUNKOCZI,A.HARONITI,E.LONGMAN,F.NIESEN,M.SOUNDARARAJAN,L.BALL,F.VON DELFT,D.DOYLE,C.ARROWSMITH,A.EDWARDS,M.SUNDSTROM

Site:

Oxford

ICM Datapack:

ICM Datapack

2BT2

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.

RGS16 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.

RGS16 is abundant in the retina (Snow et al, 1998) and also highly expressed in the lung (Larminie et al, 2004). At a cellular level RGS16 along with other RGS proteins is expressed in T lymphocytes (Beading et al, 1999). These cells play a central role in the inflammatory response of asthma. T cell activation and migration to an inflammatory stimulus in the lung appears to be regulated by RGS16 (Lippert et al, 2003). Expression of RGS16 is also increased by TNFa (Patten et al, 2003). As TNFa is also known to participates in inflammatory disorders this implies that RGS16 may function as a regulator of the inflammatory response.

Materials and Methods

RGS16

PDB:2BT2

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:RGS 16-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated thrombin protease site: MHHHHHHSSGVDLGTENLYFQ*SH
Host:BL-21(DE3)

Construct

Prelude:
Sequence:
Vector:pLIC-SGC1

Growth

Medium:
Antibiotics:
Procedure:The construct was transformed into E. coli BL21(DE3 ) cells and grown as a 1mL Terrific Broth + 100 µg/mL ampicillin culture overnight at 37°C with shaking at 700 rpm. This was used to prepare a glycerol stock by adding glycerol to a final concentration of 15 % before storage at -80°C. Using the frozen glycerol stock a 1 litre Terrific Broth + 100 µg/mL ampicillin culture was inoculated. The cultures were induced at approximately 2.3 OD600 with 1.0 mM IPTG at 25 degrees for 4 hours (shaking at 200 rpm) and harvested on the same day. The pellets were frozen at -80°C.

Purification

Procedure

Extraction

Procedure
Cell Lysis - Lysis Buffer: 50 mM Tris/ HCl pH 7.5, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP. 1 tablet protein inhibitor in 25 mL Lysis Buffer was added to the 1L growth pellet. Total vol: 40 mL (estimate). Cell breakage: 5 passes through the Emulsiflex C5 high pressure homogeniser, brief sonication on ice (10s bursts). Precipitation of DNA: Addition of PEI (final conc. 0.15%), followed by incubation for 10 min. at 4°C. Total vol: 50 mL (estimate). Removal of cell debris: After centrifugation for 30 mins at 17000 rpm and 4°C the pellet was discarded, and the supernatant filtered (0.45 µm).
Concentration:
Ligand
MassSpec:
Crystallization:Crystals grew from a 1:2 ratio mix of RGS16A-to-reservoir (26 % PEG3350, 0.1 M ammonium acetate, 0.1 M Bis-Tris pH 5.5 ).
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Beadling, C., Druey, K. M., Richter, G., Kehrl, J. H., Smith, K. A. (1999). Regulators of G protein signaling exhibit distinct patterns of gene expression and target G protein specificity in human lymphocytes. J. Immunol. 162: 2677-2682.

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.

Larminie, C., Murdock, P., Walhin, J. P., Duckworth, M., Blumer, K. J., Scheideler, M. A., Garnier, M. (2004). Selective expression of regulators of G-protein signaling (RGS) in the human central nervous system.
Brain Res. Mol. Brain Res. 122: 24-34.

Lippert, E., Yowe, D. L., Gonzalo, J. A., Justice, J. P., Webster, J. M., Fedyk, E. R., Hodge, M., Miller, C., Gutierrez-Ramos, J. C., Borrego, F., Keane-Myers, A., Druey, K. M. (2003) Role of regulator of G protein signaling 16 in inflammation-induced T lymphocyte migration and activation. J. Immunol. 17:1542-1555.

Patten, M., Stube, S., Thoma, B., Wieland, T.(2003). Interleukin-1beta mediates endotoxin- and tumor necrosis factor alpha-induced RGS16 protein expression in cultured cardiac myocytes.Naunyn Schmiedebergs Arch Pharmacol. 368: 360-365.

Snow, B. E., Antonio, L., Suggs, S., Siderovski, D. P. (1998). Cloning of a retinally abundant regulator of G-protein signaling (RGS-r/RGS16): genomic structure and chromosomal localization of the human gene.
Gene. 206: 247-253.