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Title: Activation of phospholipase D by G protein-coupled receptors
Author: McCulloch, Derek A.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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The activation of phospholipase D (PLD) by the G protein-coupled receptor (GPCR) family was investigated with special attention paid to the interaction of receptors with small G proteins such as ADP-ribosylation factor (ARF) and RhoA. Agonist-stimulated activation of PLD by the M3 muscarinic, B2 bradykinin and H1 histamine receptors was sensitive to brefeldin A (BFA), an inhibitor of guanine-nucleotide exchange on ARF. In contrast the thrombin and thromboxane A2 receptors stimulated a PLD response insensitive to BFA. The Rho inhibitor C3 exoenzyme from Clostridium botulinum and a negative functional construct of RhoA markedly reduced PLD activity stimulated by the M3 muscarinic but not the thrombin receptor. The receptors investigated which couple to PLD by a mechanism involving ARF all contain in their seventh transmembrane helix (TM7), the amino acid sequence AsnProXXTyr (where X is any amino acid). In contrast, the receptors such as thrombin which activate PLD in an ARF-independent manner have an AspProXXTyr motif. The importance of this highly conserved motif was further indicated by studies on the mouse gonadotropin-releasing hormone (GnRH) receptor which contains the AspProXXTyr sequence and a mutant form of the GnRH receptor where the aspartate at position 318 is replaced with an asparagine thereby restoring the AsnProXXTyr motif present in the majority of group I GPCRs. The wild-type GnRH receptor stimulated PLD in a BFA-insensitive manner, while the Asn318 mutant GnRH form gained BFA-sensitivity. The co-immunoprecipitation of the agonist-treated Asn318 mutant but not wild-type GnRH receptor using antibodies raised to ARF 1/3 and RhoA indicated that a direct physical association between the receptor and small G proteins occurs and appears to be dependent on the presence of the AsnProXXTyr motif.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available