Abstract
Receptor mediated stimulation of the G protein-α subunit leads to exchange of GDP for GTP, activating the protein. Spontaneous GDP release from Gα can also lead to the active state, if GTP in solution binds the nucleotide binding pocket. The purpose of this study is to evaluate the molecular determinants for maintaining the spontaneous GDP release rates between two Gα subunits. Gαt has a low rate of nucleotide release, compared to Gαi1. Gαt/i1 chimeras were used to explore the molecular basis for this behavior. The C-terminal α4-helix, the N-terminal 56 residues and the Switch I/II regions of Gαt were shown to affect the low spontaneous GDP release rate in Gαt. A specific molecular contact between Asp26 and Asn191 was found in Gαt that is not present in Gαi1. In two chimeras disrupting this interaction produced an increased spontaneous GDP release; restoring the contact present in Gαt into these chimeras decreased the GDP release rate by half as compared to the original chimeras. Similarly, introduction of this contact in wild-type Gαi1 decreased the GDP release rate of Gαi1 by half. Differences in GDP release rates may reflect physiological roles these proteins play in living systems.
Published Version
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