G-protein alpha subunit interaction and guanine nucleotide dissociation inhibitor activity of the dual GoLoco motif protein PCP-2 (Purkinje cell protein-2).
Purkinje cell protein-2 (PCP-2; L7/GPSM4) is a GoLoco motif-containing protein that is specifically expressed in Purkinje and retinal ON bipolar cells. An alternative splice variant of PCP-2 has recently been isolated which contains two GoLoco motifs. Although the second GoLoco motif (GL2) of PCP-2 has been reported to interact with Galpha-subunits, a complete biochemical analysis of each individual motif of PCP-2 has not been performed. We demonstrate that the first GoLoco motif (GL1) of PCP-2 is equipotent as a guanine nucleotide dissociation inhibitor (GDI) towards Galphai1 and Galphai2, while it has sevenfold lower GDI activity for Galphai3 and greater than 20-fold lower GDI activity against Galphao. In contrast we found PCP-2 GL2 to be essentially equipotent as a GDI for all Galphai subunits, but it had negligible activity toward Galphao. Using co-immunoprecipitation from COS-7 cells, we found that PCP-2 was only able to interact with Galphai1 but not Galphao nor Galpha-subunits from other families (Galphas, Galphaq, or Galpha12). Mutational analysis of a non-canonical residue (glycine 24) in human PCP-2 GL1 provided evidence for heterogeneity in mechanisms of Galphai interactions with GoLoco motifs. Collectively, the data demonstrate that PCP-2 is a comparatively weak GoLoco motif protein that exhibits highest affinity interactions and GDI activity toward Galphai1, Galphai2, and Galphai3 subunits.
Willard
Francis S
FS
Department of Pharmacology, CB# 7365, 1106 Mary Ellen Jones Building, University of North Carolina, Chapel Hill, NC 27599-7365, USA. fwillard@med.unc.edu
McCudden
Christopher R
CR
Siderovski
David P
DP
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance
D044385:GTP-Binding Protein alpha Subunits; D020730:Guanine Nucleotide Dissociation Inhibitors; D009419:Nerve Tissue Proteins; C091104:Purkinje cell protein L7