Section 10
Chapter 9,749

A cysteine-3 to serine mutation of the G-protein Gi1a abrogates functional activation by the a2A-adrenoceptor but not interactions with the bc complex

Wise, A.; Grassie, M.A.; Parenti, M.

Biochemistry (American Chemical Society) 36: 620-9


Accession: 009748873

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Pertussis toxin-resistant (C351G) and also palmitoylation-negative (C3S/C351G), myristoylation-negative (G2A/C351G) and combined acylation-negative (G2A/C3S/C351G) forms of the G-protein Gi1a were expressed in COS-7 cells along with the porcine a2A-adrenoceptor. G2A/C3S/C351G Gi1a and G2A/C351G Gi1a were largely cytosolic and failed to interact with the agonist-occupied a2A-adrenoceptor in membrane preparations. In contrast, C351G Gi1a was almost entirely particulate and the a2-adrenoceptor agonist UK14304 caused a marked stimulation of its GTPase activity and binding of [35S]GTPcS which was not prevented by pertussis toxin treatment of the cells. C3S/C351G Gi1a was present in both the particulate and cytosolic fractions but the GTPase activity of the membrane bound fraction was only slightly activated by the a2A-adrenoceptor. Coexpression of C3S/C351G Gi1a and the a2A-adrenoceptor along with b1 and c2 subunits increased the P2 membrane complement of the a subunit and increased substantially the ratio of membrane bound to cytosolic protein. However, this also failed to allow marked stimulation of high-affinity GTPase activity by the a2A-adrenoceptor despite the increased proportion of G-protein in the P2 membrane fraction. Despite the low fractional activation of C3S/C351G Gi1a by the a2A-adrenoceptor compared to C351G Gi1a, the palmitoylation-resistant G-protein caused a marked reduction in pertussis toxin-resistant, agonist (UK14304)-mediated stimulation of adenylyl cyclase activity. UK14304 caused the same degree of effect on adenylyl cyclase activity in pertussis toxin-treated cells following transfection of the same amounts of C351G Gi1a and C3S/C351G Gi1a, as both appear to act to sequester bc subunits. By contrast, neither G2A/C351G Gi1a nor G2A/C3S/C351G Gi1a resulted in effective regulation of adenylyl cyclase activity. Copyright 1997, American Chemical Society.

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