Differential G protein activation by fusion proteins between the human delta-opioid receptor and Gi1alpha/Go1alpha proteins

Moon, Hyo-Eun (2001) Differential G protein activation by fusion proteins between the human delta-opioid receptor and Gi1alpha/Go1alpha proteins. PhD thesis, University of Glasgow.

Abstract

Assessment of the functional activities of agonists acting on the human 6-opioid receptor (hDOR) which couples to G proteins of the Gi/Goalpha class usually involves the measurement of adenylyl cyclase (AC) activity. To assess the relative capacitiy of the hDOR to activate closely related G proteins, fusion proteins were constructed in which the a subunits of either Gi1 or Go1, containing point mutations to render them insensitive to the actions of pertussis toxin (PTx), were linked in frame with the C- terminus of the receptor. Following transient and stable expression in HEK293 cells both constructs bound the antagonist [3H] naltrindole with high affinity. D-ala2, D- leu5 enkephalin [DADLE] effectively inhibited forskolin-stimulated AC activity in intact cells in a concentration-dependent but PTx-insensitive manner. The high affinity GTPase activity of both constructs was also stimulated by DADLE with similar potency. However, enzyme kinetic analysis of agonist stimulation of GTPase activity demonstrated that the GTP turnover number produced in response to DADLE was more than 3 times greater for Gi1alpha than for Go1alpha. As the effect of agonist in both cases was to increase Vmax without increasing the observed Km, for GTP this is consistent with receptor promoting greater guanine nucleotide exchange on, and thus activation of, Gi1alpha compared to Go1alpha. An equivalent fusion protein between the human mu-opioid receptor-1 and Gi1alpha produced a similar DADLE-induced GTP turnover number as the hDOR-Gi1alpha fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of Gi1alpha. In addition, I have investigated the characteristics of the ternary complex of agonist-GPCR-G protein using high affinity agonist binding studies with the fusion proteins. GDP reduced the binding of the agonist [3H] DADLE but not the antagonist [3H] naltrindole to both the receptor alone and all the hDOR-Gi1alpha (Xaa351) 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 displayed similar association 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 the more hydrophobic residue 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 was 2 fold greater with the isoleucine351 containing fusion protein and the potency of DADLE was 7 fold greater with the isoleucine351 fusion protein than for the version containing glycine. These results demonstrate that the stability of the ternary complex between hDOR, Gi1alpha 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 receptor to G protein.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Neurosciences, Molecular biology
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Milligan, Professor Graeme
• Date of Award: 2001
• Depositing User: Enlighten Team
• Unique ID: glathesis:2001-72487
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Jun 2019 11:06
• Last Modified: 11 Jul 2022 08:05
• Thesis DOI: 10.5525/gla.thesis.72487
• URI: https://theses.gla.ac.uk/id/eprint/72487

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