Human Vasoactive Intestinal Peptide Receptor 2 - GPCR extracellular domain

Target ID:

VIPR2A

Aliases:

FLJ16511VIPR2VPAC2VPCAP2R

Deposition Date:

Friday 5th February 2010

Families:

Membrane proteins

Structure type:

apo

Download Coordinates:

2X57

Authors:

A.C.W.PIKE,A.J.BARR,A.QUIGLEY,N.BURGESS BROWN,A.DE RISO,A.BULLOCK,G.BERRIDGE,J.R.C.MUNIZ,A.CHAIKAUD,M.VOLLMAR,T.KROJER,E.UGOCHUKWU,F.VON DELFT,A.EDWARDS,C.H.ARROWSMITH,J.WEIGELT,C.BOUNTRA,E.P.CARPENTER

Site:

Oxford

ICM Datapack:

ICM Datapack

2X57

tabs

Structure Details

VIPR1 and 2 belong to the class II subfamily of the seven-transmembrane (7TM) G-protein-coupled receptor (GPCR) superfamily. The Class II GPCRs are characterised by a 100 to 200 residue amino terminal extracellular domain with 3 conserved disulphide bonds. VIPR1 and 2, also termed the VPAC1 and VPAC2 receptors respectively, are 49% conserved on the amino acid level and serve as receptors for two related neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP) (Laburthe et al., 2002).

The vasoactive intestinal peptide (VIP) is a neuropeptide and endocrine hormone with wide ranging effects on the gut, brain and heart. In the digestive system VIP controls secretion of water and electrolytes into the gut, secretion of water into pancreatic juice and bile, relaxation of smooth muscle in parts of the gut and regulation of gastric acid and pepsinogen secretion. In the brain VIP acts as a neuropeptide, passing signals between neurons, particularly those associated with the Suprachiasmatic nuclei, the region responsible for the brain's timekeeping function, or circadian rhythms.
Knockout mice for the VIP receptor VIPR2 established the essential role for this receptor in controlling the maintenance of circadian rhythm and further analysis demonstrated that VIPR2 is not only essential for synchronisation of circadian pacemakers in the suprachiasmatic nuclei (SCN) but is also necessary to maintain the molecular timekeeping within individual SCN neurons and to regulate the excitability of SCN neurons (Harmar, 2003), (Maywood et al., 2007). In addition, mice lacking VIPR2 show increased delayed type hypersensitivity but depressed immediate-type hypersensitivity demonstrating the involvement of VIPR2 in regulation of T-cell function (Goetzl et al., 2001). VIPR2 also regulates insulin secretion, growth, lipolysis and male reproductive functions as demonstrated by a conditional knockout lacking exon 8-10 of VIPR2 (Asnicar et al., 2002).
VIP receptor manipulation is used in treatments for several diseases: VIPR2 agonists are used as bronchodilators in asthma and are considered for the treatment of Type II diabetes (Schmidt et al., 2001), (Pan et al., 2007). VIPR2 has been suggested as a target for antiinflammatory agents in rheumatoid arthritis (Gomariz et al., 2006). Overexpression of VIPR2 has been described in central primitive neuroectodermal tumours as well as gastrointestinal tumours. Overproduction of VIP in pancreatic tumours (VIPomas) results in chronic diarrhoea. Polymorphisms in VIPR2 may affect gastrointestinal symptoms and stereotypical behaviours in autism (Vaudry et al., 2009), (Asano et al., 2001). VIP is also involved in heart function, causing coronary vasodilation and it is therefore being investigated as a possible treatment for heart failure.

Here we describe the structure of VIPR2 extracellular domain at 2.1 Å. In common with the other class B GPCR extracellular domains the VIPR2 has an N-terminal α-helix packed against a 2-stranded β-sheet and a 3-stranded β-sheet. The structure is held together by three disulphide bonds. Although this is an unliganded structure, crystallised without peptide bound, it is known from related crystal and NMR structures that the C-terminus of the peptide hormones usually bind in the region of loops 2 and 4 (Parthier, et al., 2009). The N-terminus of the VIP would then extend into the C-terminal domain of the receptor, the seven-transmembrane GPCR domain, thus transmitting the signal across the cell membrane via the G-α GTPase to adenylate cyclase or phospholipase C.

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: (IMAGE:4178593)

SGC Construct ID: VIPR2A-c104

GenBank GI number: gi|21361557

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Coding DNA sequence:
CATATGCACCATCATCATCATCATTCTTCT
GGTGTAGATCTGGGTACCGAGAACCTGTAC
TTCCAATCCATGCGATTTCATCTGGAAATA
CAGGAGGAAGAAACAAAATGTGCAGAGCTT
CTGAGGTCTCAAACAGAAAAACACAAAGCC
TGCAGTGGCGTCTGGGACAACATCACGTGC
TGGCGGCCTGCCAATGTGGGAGAGACCGTC
ACGGTGCCCTGCCCAAAAGTCTTCAGCAAT
TTTTACAGCAAAGCAGGAAACATAAGCAAA
AACTGTACGAGTGACGGATGGTCAGAGACG
TTCCCAGATTTCGTCGATGCCTGTGGCTAC
AGCGACCCGGAGGATGAGAGCTGACAGTAA
AGGTGGATACGGATCCGAA

Tags and additions: Cleavable N-terminal His6 tag (* TEV cleave site).

Host: BL21 (DE3)R3-pRARE2 (Phage resistant strain)

Final protein sequence (small letters refer to tag sequence):
mhhhhhhssgvdltenlyfq*SMRFHLEIQ
EEETKCAELLRSQTEKHKACSGVWDNITCW
RPANVGETVTVPCPKVFSNFYSKAGNISKN
CTSDGWSETFPDFVDACGYSDPEDES

Growth medium, induction protocol: 5 ml from an overnight culture containing 50 µg/ml kanamycin & 34 µg/ml chloramphenicol was used to inoculate each 1 L culture of LB. Cultures were grown at 37 °C until the OD₆₀₀ reached ~1.8 then the temperature was adjusted to 20 °C. Expression was induced using 0.5 mM IPTG added at an OD₆₀₀ of ~2.0 and the cells were grown overnight. The cells were collected by centrifugation, and the pellet was resuspended in binding buffer and frozen. For selenomethionine labeling of protein, 50 ml of an overnight culture was washed with methionine minus medium (Molecular Dimensions Ltd.) and used to inoculate 8 L of methionine minus medium supplemented with 40 mg / L of L-selenomethionine (Acros Organics) containing 50 µg/ml kanamycin & 34 µg/ml chloramphenicol. Protein expression was induced as above for the native protein.

Binding buffer: 50 mM HEPES pH 7.5; 500 mM NaCl.

Extraction buffer, extraction method: Frozen pellets were thawed and cells lysed with 3 passes through a C3 high pressure cell disrupter (Avestin). The lysate was centrifuged at 17,000 rpm for 30 minutes and the pellet was retained for purification. The pellet was solubilized in solubilization buffer using a Teflon homogenizer followed by mixing on a rotating platform for 1 hour at room temperature. The solubilized lysate was centrifuged at 17,000 rpm for 30 minutes and the supernatant collected for purification.

Column 1: Ni-affinity. Nickel-IDA (Sterogene), 5 ml of 50 % slurry in 1.5 x 10 cm column, washed with solubilization buffer.

Buffers:
Solubilization buffer: 25 mM HEPES, pH 7.5; 8 M Urea; 1 M NaCl; 5 mM imidazole and 0.5 mM TCEP
Wash buffer: 25 mM HEPES, pH 7.5; 8 M Urea; 1 M NaCl; 30 mM imidazole and 0.5 mM TCEP
Elution buffer: 25 mM HEPES, pH 7.5; 8 M Urea; 1 M NaCl; 50 to 400 mM imidazole and 0.5 mM TCEP

Procedure: The solubilized lysate was loaded by gravity flow on the Ni-IDA column. The column was then washed with 100 ml solubilization buffer and 100 ml of wash buffer. The protein was eluted by applying 10 ml portions of elution buffer with increasing concentration of imidazole (50 mM, 150, 250 and 400 mM); fractions were collected until essentially all protein was eluted.

Protein Refolding: Eluted protein was transferred to dialysis tubing (3 kDa cut-off) and dialysed against 2 L of refolding buffer overnight at 4°C, followed by a further 8 hour dialysis in fresh refolding buffer. The protein was then dialysed overnight against 1 L of dialysis buffer overnight at 4°C.

Buffers:
Refolding buffer: 50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 0.4 M arginine, 0.2 mM reduced glutathione, 0.2 mM oxidised glutathione and 5 mM EDTA
Dialysis Buffer: The protein was then dialysed overnight against 50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol

Column 2: Size Exclusion Chromatography. Superdex S75 16/60 HiLoad

Buffers: 50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol

Procedure: The protein was concentrated and applied to an S75 16/60 HiLoad gel filtration column equilibrated in 50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol using an ÄKTAxpress system.

Mass spectrometry characterization: LC-ESI MS TOF analysis indicated the purified protein had a mass of 13241.7 Da. The measured mass is in accordance with the expected native mass (13154 Da) with the incorporation of two selenomethionines and the loss of 6 Da as a result of formation of 3 disulphide bonds.

Protein concentration: Protein was concentrated to 25 mg/ml using a 3 kDa cut-off concentrator (Amicon).

Crystallization: Crystals were grown at 20 °C in 150 nl sitting drops comprising a 1:1 ratio of protein (25mg/ml) to reservoir solution containing 40% PEG 300; 0.1 M citrate/PO4 pH 4.2

Data Collection: Crystals were vitrified directly in liquid nitrogen. "Native" and SAD datasets were collected from crystals grown with selenomethionine-substituted protein on beamline I03 at the Diamond Light Source. Phasing information was obtained from a 2.9 Å Se-SAD dataset collected at the selenomethionine peak wavelength (λ = 0.9799 Å). High resolution "native" data, extending to 2.1 Å resolution, were obtained from similarly substituted crystals collected at a wavelength below the peak (λ = 0.9809 Å).

References

Asano, E., Kuivaniemi, H., Huq, A.H., Tromp, G., Behen, M., Rothermel, R., Herron, J., and Chugani, D.C. (2001). A study of novel polymorphisms in the upstream region of vasoactive intestinal peptide receptor type 2 gene in autism. J Child Neurol 16, 357-363.
Asnicar, M.A., Koster, A., Heiman, M.L., Tinsley, F., Smith, D.P., Galbreath, E., Fox, N., Ma, Y.L., Blum, W.F., and Hsiung, H.M. (2002). Vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor 2 deficiency in mice results in growth retardation and increased basal metabolic rate. Endocrinology 143, 3994-4006.
Goetzl, E.J., Voice, J.K., Shen, S., Dorsam, G., Kong, Y., West, K.M., Morrison, C.F., and Harmar, A.J. (2001). Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC(2) receptor for vasoactive intestinal peptide. Proc Natl Acad Sci U S A 98, 13854-13859.
Gomariz, R.P., Juarranz, Y., Abad, C., Arranz, A., Leceta, J., and Martinez, C. (2006). VIP-PACAP system in immunity: new insights for multitarget therapy. Ann N Y Acad Sci 1070, 51-74.
Harmar, A.J. (2003). An essential role for peptidergic signalling in the control of circadian rhythms in the suprachiasmatic nuclei. J Neuroendocrinol 15, 335-338.
Laburthe, M., Couvineau, A., and Marie, J.C. (2002). VPAC receptors for VIP and PACAP. Receptors Channels 8, 137-153.
Maywood, E.S., O'Neill, J.S., Chesham, J.E., and Hastings, M.H. (2007). Minireview: The circadian clockwork of the suprachiasmatic nuclei--analysis of a cellular oscillator that drives endocrine rhythms. Endocrinology 148, 5624-5634.
Pan, C.Q., Li, F., Tom, I., Wang, W., Dumas, M., Froland, W., Yung, S.L., Li, Y., Roczniak, S., Claus, T.H., et al. (2007). Engineering novel VPAC2-selective agonists with improved stability and glucose-lowering activity in vivo. J Pharmacol Exp Ther 320, 900-906.
Schmidt, D.T., Ruhlmann, E., Waldeck, B., Branscheid, D., Luts, A., Sundler, F., and Rabe, K.F. (2001). The effect of the vasoactive intestinal polypeptide agonist Ro 25-1553 on induced tone in isolated human airways and pulmonary artery. Naunyn Schmiedebergs Arch Pharmacol 364, 314-320.
Vaudry, D., Falluel-Morel, A., Bourgault, S., Basille, M., Burel, D., Wurtz, O., Fournier, A., Chow, B.K., Hashimoto, H., Galas, L., et al. (2009). Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery. Pharmacol Rev 61, 283-357.
Parthier, C., Reedtz-Runge, S., Rudolph, R. and Stubbs, M.T.,(2009) Passing the baton in class B GPCRs: peptide hormone activation via helix induction? TIBS, 34. 6, 303-310.