Differential G-alpha interaction capacities of the GoLoco motifs in Rap GTPase activating proteins.
GoLoco motif proteins act as guanine nucleotide dissociation inhibitors (GDIs) for G-protein alpha subunits of the adenylyl cyclase-inhibitory (Galpha(i/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 Galpha 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 Galpha 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 Galpha(i1). In contrast, the GoLoco motif of Rap1GAP1b, which is canonical in primary sequence, was found to interact with Galpha(i1).GDP.
Willard
Francis S
FS
Department of Pharmacology, CB# 7365, 1106 Mary Ellen Jones Building, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365 USA. fwillard@med.unc.edu
Low
Aaron B
AB
McCudden
Christopher R
CR
Siderovski
David P
DP
D020816:Amino Acid Motifs; D001665:Binding Sites; D017124:Conserved Sequence; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D044385:GTP-Binding Protein alpha Subunits; D020690:GTPase-Activating Proteins; C500320:RAP1GAP2 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go