Elsevier

Neuropharmacology

Volume 41, Issue 3, September 2001, Pages 321-330
Neuropharmacology

Control of the efficiency of agonist-induced information transfer and stability of the ternary complex containing the δ opioid receptor and the α subunit of Gi1 by mutation of a receptor/G protein contact interface

https://doi.org/10.1016/S0028-3908(01)00076-4Get rights and content

Abstract

Fusion proteins were constructed between the δ opioid receptor and forms of the α subunit of Gi1 in which cysteine351 was mutated to a range of amino acids. GDP reduced the binding of the agonist [3H]DADLE but not the antagonist [3H]naltrindole to both the receptor alone and all the δ opioid receptor–Cys351XaaGi1α fusion proteins. For the fusion proteins the pEC50 for GDP was strongly correlated with the n-octanol/H2O partition co-efficient of G protein residue351. Fusion proteins in which this residue was either isoleucine or glycine had similar observed binding kinetics for [3H]DADLE. However, the rate of dissociation of [3H]DADLE was substantially greater for the glycine-containing fusion protein than that containing isoleucine, indicating that more hydrophobic residues imbued greater stability to the agonist–receptor–G protein ternary complex. This resulted in a higher affinity of binding of [3H]DADLE to the fusion protein containing isoleucine351. In expectation with the binding data, maximal DADLE-stimulated GTP hydrolysis by the isoleucine351-containing fusion protein was two-fold greater and the potency of DADLE seven-fold higher than for the version containing glycine.

These results demonstrate that the stability of the ternary complex between δ opioid receptor, Gi1α and an agonist (but not antagonist) ligand is dependent upon the nature of residue351 of the G protein and that this determines the effectiveness of information flow from the receptor to the G protein.

Introduction

Evidence from a wide range of experimental approaches has indicated that the C-terminal region of G protein α subunits plays a key role in the selectivity of interactions with members of the family of G protein-coupled receptors (GPCRs) (Hamm, 1998, Wess, 1998, Gether and Kobilka, 1998, Schoneberg et al., 1999). These include that the unc mutation of Gsα, which results from an Arg/Pro alteration 6 amino acids from the C-terminus, prevents productive interactions with GPCRs (Sullivan et al., 1987), the capacity of antisera directed towards the C-terminal region of G proteins to interfere selectively with activation by GPCRs (Milligan, 1994), the widespread use of chimeric G proteins, in which the extreme C-terminal region of G proteins are exchanged, to alter GPCR coupling specificity (Milligan and Rees, 1999) and detection of conformational changes in this region associated with G protein activation (Yang et al., 1999). Furthermore, pertussis toxin treatment prevents effective interactions of GPCRs with Gi-family G proteins by catalysing the addition of ADP-ribose to a Cys residue conserved between these G proteins and located 4 amino acids from the C-terminus (Milligan, 1988).

Because the Gi-family G proteins are widely expressed and appropriate null backgrounds are not available, many studies have employed mutants of these proteins in which the pertussis toxin-sensitive Cys has been altered (Senogles, 1994, Chuprun et al., 1997, Yamaguchi et al., 1997, Wise et al., 1997b, Leaney and Tinker, 2000). This has allowed the elimination of potential interactions of GPCRs with endogenously expressed forms of Gi by prior pertussis toxin treatment and analysis of interactions by reconstitution of function provided by the mutated G protein. In studies with the α2A-adrenoceptor, Bahia et al. (1998) co-expressed this GPCR along with forms of Gi1α in which the pertussis toxin-sensitive Cys was replaced by all of the other naturally occuring amino acids. They observed a wide range of capacity of the agonist-occupied receptor to activate the modified forms of the G protein. However, certain mutated G proteins were poorly expressed and this posed difficulties for quantitative analysis (Bahia et al., 1998). To overcome this problem a number of recent studies have utilised fusion proteins in which single open reading frames containing both GPCR and G protein function are produced. These have provided a range of insights into GPCR-G protein interactions and selectivity (see Seifert et al., 1999b, Milligan, 2000 for reviews). Herein, fusion proteins between the human δ opioid receptor (DOR) and a range of modified forms of Gi1α have been employed to explore the effect of G protein mutation on agonist–GPCR–G protein ternary complex formation and dissociation and the stability of this ternary complex in GPCR to G protein information transfer.

Section snippets

Methods

All materials for tissue culture were supplied by Life Technologies Inc (Paisley, Strathclyde, Scotland, UK). [3H]naltrindole (33 Ci/mmol), [3H]DADLE (55.3 Ci/mmol) and [γ-32P] GTP (30 Ci/mmol) were obtained from NEN life science products. Pertussis toxin (240 μg/ml) was purchased from Speywood. All other chemicals were from Sigma or Fisons plc (Loughborough, UK) and were of the highest purity available. Oligonucleotides were synthesised by Cruachem Limited (Glasgow, UK).

Results

Agonist-induced exchange of GTP for GDP on a G protein is accepted as the rate-limiting step of the GTP exchange and hydrolysis cycle and reflects enhanced dissociation of GDP from the nucleotide binding site (Gilman, 1987). Thus, agonist stimulation of subsequent GTPase activity provides a direct monitor of G protein activation by the agonist-occupied receptor (Gierschik et al., 1994). As addition of agonist reduces the affinity of the G protein for GDP, then increasing concentrations of GDP

Discussion

The current studies demonstrate that the identity of residue351 of Gi1α alters both the effectiveness of agonist-induced activation of the G protein by the DOR and determines the stability of a ternary complex between agonist, DOR and Gi1α by altering its rate of association and dissociation. These two features are likely to be inherently related as effective maintenance of the ternary complex is required to allow agonist-induced information transfer between the partner proteins.

The extreme

Acknowledgements

H.-E. Moon thanks the Institute of Biomedical and Life Sciences, University of Glasgow for a studentship. Financial support for this work was provided by the Medical Research Council (UK).

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