G-protein specificity


G-proteins are central mediators of the signal transduction process, conveying information from agonist activated receptor to intracellular effector. However, it is unclear whether G-proteins function in a specific manner, or are promiscuous and able to interact with a wide variety of transmembrane receptors and effectors. In an attempt to analyse G-protein specificity, the interaction of receptors with G-proteins in the neuroblastoma x glioma NG108-15 cell line was examined. As a means of identifying individual members of the G-protein family, a series of antipeptide antisera were generated against synthetic peptides corresponding to the C-terminal region of the various G-protein -subunits. These antisera were demonstrated to be specific in their ability to recognise individual members of the G-protein family and thus allowed the identification of the pertussis toxin sensitive G-proteins; Gi2, Gi3, and Go in the NG108-15 cell line. In addition, two forms of the cholera toxin sensitive G-protein, Gs were expressed. However it was not possible to detect expression of Gil. NG108-15 cells express -oploid receptors which function to inhibit adenylyl cyclase in a manner attenuated by prior treatment with pertussis toxin, and a poorly defined growth factor receptor which does not functionally interact with adenylyl cyclase. In membranes derived from NG108-15 cells, agonist activation of the both the -opioid receptors and growth factor receptors by DADLE and foetal calf serum respectively, stimulated GTPase activity through activation of separate species of pertussis toxin sensitive G-protein, as assessed by additivity experiments. This demonstrated that tight regulation of receptor-G-protein coupling occurs in membrane systems. In an attempt to determine which pertussis toxin sensitive G-protein(s) is activated by the -opioid receptor, and thus ascertain which G-protein functions to inhibit adenylyl cyclase, a variety of approaches were adopted. Firstly, although 'Gi' is classically a substrate for pertussis toxin, it is possible to catalyse the ADP-ribosylation of 'Gi' with cholera toxin, when suitable conditions are employed, namely the absence of added guanine nucleotide. In NG108-15 cell membranes, cholera toxin catalysed ADP-ribosylation of 'Gi' was stimulated in a dose-dependent manner by the addition of the -opioid agonist, DADLE, thus providing a means of identifying the G-protein which interacts with the -opioid receptor. Secondly, the C-terminal region of the G-protein ?-subunit is postulated to be the domain of interaction with receptor. In an attempt to block interaction of the -opioid receptor with the G-protein with which the receptor interacts, NG108-15 cell membranes were preincubated with IgG fractions isolated from the various G-protein specific antisera. Functional uncoupling of the -opioid receptor from both GTPase stimulation and inhibition of adenylyl cyclase was achieved by preincubation with an IgG fraction isolated from antiserum AS7, an antiserum which specifically recognizes Gi2 in this cell line. Preincubation with IgG fractions isolated from either Gi3, Go or Gs specific antisera were ineffective. Preincubation with an IgG fraction isolated from preimmune serum was also ineffective. Thirdly, pertussis toxin catalysed ADP-ribosylation of Gi attenuates productive coupling between receptor and Gi, leaving the receptor in a state with lowered affinity for agonist. Preincubation of NG108-15 cell membranes with an IgG fraction isoiated from antiserum AS7 produced the same functional consequence as pertussis toxin pretreatment. As before, preincubation with IgG fractions derived from antisera specific for Gi3, Go, Gs or preimmune serum were ineffective. These data demonstrate that in cell membranes, receptors are specific in their ability to activate G-proteins, the -opioid receptor functions through activation of Gi2, and that Gi2 must therefore be the G-protein which mediates inhibition of adenylyl cyclase In NG108-15 cell membranes

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oaioai:theses.gla.ac.uk:72853Last time updated on 7/9/2019View original full text link

This paper was published in Glasgow Theses Service.

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