The role of the C-terminus in defining the efficiency and specificity of G-protein coupling

Bahia, Daljit Singh (1999) The role of the C-terminus in defining the efficiency and specificity of G-protein coupling. PhD thesis, University of Glasgow.

Full text available as:
[img]
Preview
PDF
Download (7MB) | Preview

Abstract

In combination with conferring resistance to ADP-ribosylation by Pertussis toxin, the substitution of a conserved cysteine residue (C351) four amino acids from the C-terminus of Gialpha1 has been shown to modulate the efficiency of coupling to the α2A adrenoceptor. Investigation of this phenomenon through systematic substitution of this cysteine residue for all other amino acids highlighted a relationship between the hydrophobicity of the substituted residue and the capacity of the a-subunit to functionally couple to the α2A adrenoceptor. From the results of this investigation, it was noted that wild type Giα1 did not display optimal coupling at this receptor. Relative to wild type Giα1, coupling was enhanced by the substitution of a more hydrophobic residue at position C351, but diminished upon the substitution of a more hydrophilic residue. In contrast to this, substitution of proline or a charged residue at this position essentially attenuated functional coupling with the α2A adrenoceptor. Similarly, pEC50 values of the mutants also showed a high degree of correlation with the hydrophobic nature of the substituted residue, with more hydrophobic residues reducing pEC50 values and more hydrophilic residues increasing pEC50 values respectively relative to wild type Giαl. This change in coupling efficiency could not be attributed to a change in the affinity for nucleotides at the a-subunit or to a change in the rate of basal guanine nucleotide exchange. These data indicate that functional coupling of the Giαl subunit to the α2A adrenoceptor is in part modulated by the physiochemical properties of residue 351 and that a relationship exists between the hydrophobicity of residue 351 and the capacity of the α-subunit to functionally couple to the α2A adrenoceptor. A series of fusion constructs composed of the α2A adrenoceptor covalently linked to selected Giαl C351 mutants were used to assess the effects of substituting residue C351 in Giαl on agonist intrinsic activity at the α2A adrenoceptor. The agonist UK 14304 was shown to elicit a spectrum of responses at the fusion constructs, closely mirroring the order of coupling efficiency previously determined in the separately expressed components. While UK14304 essentially acted as a full agonist compared to adrenaline at the fusion construct composed of wild type Giαl (C351 Giαl), it was shown to act as a partial agonist at an equivalent construct containing a glycine residue at position 351 in the Giαl moiety. In contrast to this, relative to the wild type Giαl fusion, UK14304 displayed greater relative intrinsic activity at the fusion construct containing an isoleucine residue at position 351 in the Giαl moiety. Analysis of a more extensive range of partial agonists demonstrated that the order of agonist relative intrinsic activity at the respective fusion constructs was conserved regardless of the agonist assayed. This discrepancy of intrinsic activity at the fusion proteins could not be attributed to a change in the pharmacological profile of the receptor moiety or to a modification of its affinity for agonist. These data indicate that the intrinsic activity of partial agonists, relative to adrenaline, can be modulated by the physiochemical properties of residue 351 in the Giαl moiety. The capacity of a series of chimeras containing substitutions of the last 6 C- terminal residues of Giαl for those of Gsα, Gqα and G16α were assessed for functional coupling with a range of non Gi-linked receptors. Functional coupling was demonstrated at the Gi/Gs chimera with the V2 vasopressin receptor and α2 adrenoceptor. Similarly, the Gi/Gq chimera was shown to functionally couple with the P2Y4, and TRH, receptors. No functional coupling was detected for the Gi/G16 chimera. The inability of the Gi/G16 chimera to functionally couple to any of the receptors analysed was independent from its capacity to basally exchange guanine nucleotides, which was shown to be unchanged relative to the Gi/Gs, Gi/Gq chimeras and wild type Giαl. The substitution of the 6 C-terminal residues of Giαl for those of Gsα also conferred resistance to ADP-ribosylation by both pertussis and cholera toxins. This was demonstrated by functional coupling of the Gi/Gs chimera to a FLAG(TM) -tagged version of the IP prostanoid receptor following treatment with both bacterial toxins. The GiαlGsα chimera was seen to couple more efficiently at the IP prostanoid receptor in the presence of these toxins than in non toxin treated samples, indicating that coupling efficiency of this chimera at the IP prostanoid receptor was not optimal. These findings indicate that the C-terminus of the G-protein α-subunit is an important determinant in defining G-protein/receptor coupling and that additional determinants, not present in the C-terminus of the α-subunit, are required for optimal coupling efficiency.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Graeme Milligan
Keywords: Molecular biology
Date of Award: 1999
Depositing User: Enlighten Team
Unique ID: glathesis:1999-71285
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 10 May 2019 10:49
URI: http://theses.gla.ac.uk/id/eprint/71285

Actions (login required)

View Item View Item

Downloads

Downloads per month over past year