GoLoco motif proteins act as guanine nucleotide dissociation inhibitors (GDIs) for G-protein alpha subunits of the adenylyl cyclase-inhibitory (Gαi/o) class. Rap1GAP2 is a newly identified GoLoco motif- and RapGAP domain-containing protein, and thus is considered a potential integrator of heterotrimeric and monomeric GTPase signaling. Primary sequence analysis indicated that the Rap1GAP2 GoLoco motif contains a lysine (Lys-75), rather than an arginine, at the crucial residue responsible for binding the alpha and beta phosphates of GDP and exerting GDI activity. To determine the functional outcome of this sequence variation we conducted a biophysical analysis of the human Rap1GAP2b/c GoLoco motif. We found that human Rap1GAP2b/c was deficient in GDI activity and Gα interaction capability. Mutation of lysine-75 to arginine could not regain functional activity of the Rap1GAP2b/c GoLoco motif. Thus, the Rap1GAP2b/c GoLoco motif can be classed as inactive towards Gα subunits. We also found that the Rap1GAP1a GoLoco motif, which lacks seven N-terminal amino acid residues present in canonical GoLoco motifs, does not interact with Gαi1. In contrast, the GoLoco motif of Rap1GAP1b, which is canonical in primary sequence, was found to interact with Gαi1·GDP.
- GoLoco motif
- Heterotrimeric G-protein