ACCELERATED COMMUNICATION Modulation of Relative Intrinsic Activity of Agonists at the Alpha-2A Adrenoceptor by Mutation of Residue 351 of G Protein Gi1a

Compared with epinephrine, the relative intrinsic activity of a series of partial agonists to activate fusion proteins between the porcine alpha-2A adrenoceptor and the a-subunit of Gi1 was reduced after a single-point mutation (CysGly) in the G protein. Although UK14304 was close to a full agonist at the fusion construct containing wild-type (Cys)Gi1a, it was a partial agonist at that containing GlyGi1a. Moreover, although clonidine functioned as a good partial agonist to activate the fusion protein containing CysGi1a, it was essentially an antagonist at the GlyGi1a-containing fusion protein. By contrast, incorporation of IleGi1a into the fusion protein resulted in all partial agonists displaying higher intrinsic activity relative to epinephrine to activate this fusion protein than the one containing the wild-type G protein sequence. This is the first demonstration that the relative intrinsic activity of a series of agonists can be modified by a point mutation in a G protein rather than a receptor and indicates that the nature of a key contact site between a G protein and a receptor can selectively regulate partial agonist function. We provide a model for this based on the hydrophobicity of a key receptor-G protein a-subunit interaction interface. Ligand efficacy and intrinsic activity are key concepts in pharmacology (Stephenson, 1956; Hoyer and Boddeke, 1993; Clarke and Bond, 1998). They are usually equated simply with the “strength” of an agonist ligand to transmit signal after binding to a receptor. However, a molecular understanding of the basis of these parameters would provide novel insights into the conformational alterations induced by agonist binding to G protein-coupled receptors (GPCRs) that result in G protein activation. It is particularly interesting in this regard that the relative intrinsic activity of partial agonists at the beta-2 adrenoceptor has been noted to be increased after mutations of this GPCR that result in degrees of agonist-independent signal transduction (Lefkowitz et al., 1993; Samama et al., 1993). Such mutations are generically described as constitutively active mutations (CAMs) (Lefkowitz et al., 1993; Samama et al., 1993). Relative intrinsic activity can be measured at a range of points in a signaling cascade. However, due to cross-talk between pathways and varying levels of amplification throughout such cascades, differences in levels of expression of the GPCR and altered GPCR/G protein expression ratios can result in variations in this parameter when using distal points for analysis (Whaley et al., 1994; MacEwan et al., 1995). Therefore, a proximal assay point such as ligandinduced G protein activation provides a highly appropriate level for such measurements. We constructed a series of fusion proteins between the porcine alpha-2A adrenoceptor and the a-subunit of the G protein Gi1 (Wise and Milligan, 1997; Wise et al., 1997a,b; Burt et al., 1998). Because their construction defines that the ratio of expressed GPCR to G protein is always maintained at 1:1 and agonist function can be measured as activation of the GTPase activity of the G protein within the fusion construct, these have particular value in assessing relative intrinsic activity of a series of agonists (Wise et al., 1997a). We recently examined the effectiveness of a series of agonists after transient expression of an alpha-2A adrenoceptor-Gi1a fusion This work was supported by the Medical Research Council and the Biotechnology and Biosciences Research Council. ABBREVIATIONS: GPCR, G protein-coupled receptor; CAM, constitutively active mutant. 0026-895X/99/020195-07$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 55:195–201 (1999). 195 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from protein that was rendered insensitive to the actions of pertussis toxin by mutation of Cys of the G protein to Gly (Wise et al., 1997a). We were surprised to note that a number of alpha-2A adrenoceptor partial agonists, including clonidine, functioned more poorly compared with epinephrine than might have been expected. To understand the basis for these observations, we constructed further alpha-2A adrenoceptor-Gi1a fusion proteins in which the only difference was the nature and identity of the amino acid at residue 351 of the G protein sequence. We note that partial agonists vary in intrinsic activity relative to epinephrine with the identity of this amino acid. All partial agonists display reduced relative intrinsic activity at alpha-2A adrenoceptor-GlyGi1a compared with alpha-2A adrenoceptor wild-type (Cys)Gi1a, whereas they all display enhanced relative intrinsic activity to stimulate alpha-2A adrenoceptor-IleGi1a. We provide a model for this based on the hydrophobicity of a key GPCR-G protein a-subunit interaction interface. Materials and Methods Materials. All materials for tissue culture were supplied by Life Technologies, Inc. (Paisley, Strathclyde, Scotland). [H]RS-79948197 (90 Ci/mmol) was purchased from Amersham International (Buckinghamshire, U.K.). [g-P]GTP (30 Ci/mmol) was obtained from DuPont-New England Nuclear (Boston, MA). Pertussis toxin (240 mg/ml) and all other basic chemicals were purchased from Sigma (Poole, Dorset, U.K.) or Boehringer-Mannheim (Mannhein, Germany) and were of the highest purity available. Reagents for molecular biological manipulation were obtained from Promega