Sci. Signal., 27 March 2012
Biochemistry GAINing Autoproteolytic Activity
Annalisa M. VanHook
Science Signaling, AAAS, Washington, DC 20005, USA
The extracellular domains of PKD1, a member of the polycystin family of proteins implicated in polycystic kidney disease (PKD), and the G protein–coupled receptors (GPCRs) that mediate cell adhesion undergo autoproteolytic cleavage in a ~40–amino acid GPS (GPCR proteolytic site) motif that is located between the large extracellular domain and the first transmembrane helix. The mechanism of cleavage is not known because the GPS motif is predicted to be too short to constitute a catalytically active autoproteolytic domain. Structural and functional analyses of two adhesion GPCRs by Araç et al. revealed that the GPS motif is part of a larger conserved domain that is necessary and sufficient for autoproteolysis. Structures of ~400 amino acids of the extracellular domains of rat CL1 (calcium-independent receptor of α-latrotoxin 1, also called CIRL1) and human brain angiogenesis inhibitor 3 (BAI3) produced in and isolated from cultured insect cells revealed that the GPS motif was part of a ~320-residue domain that the authors dubbed the GAIN (GPCR autoproteolysis-inducing) domain, which formed a separate structural unit from the ~70-residue hormone receptor domain located N-terminal to the GAIN domain. Immunoblotting of lysates from human embryonic kidney (HEK) 293 cells transfected with expression constructs encoding portions of the GAIN domain of CL1 demonstrated that the entire GAIN domain of CL1 was required for autoproteolysis; neither the GPS motif alone nor the GAIN domain in which the GPS motif had been deleted exhibited autoproteolysis. The GAIN domain of another cell adhesion GPCR, GPR56, also exhibited autoproteolysis in the assay. For both CL1 and CL3, the cleaved extracellular domain remained associated with the rest of the protein, suggesting that the GAIN domain structure remained stable even after hydrolysis, thus preventing the cleavage products from dissociating. Sequence analysis indicated that all members of the cell adhesion family of GPCRs and PKD homologs contain a GAIN domain. Several mutations that are associated with autosomal-dominant PKD (ADPKD) map to the GAIN domain of PKD1, and mutations associated with cancer map to the GAIN domains of CL1, CL3, and BAI3. Whereas wild-type human PKD1 exhibited autoproteolysis in HEK293 cells, nine mutant proteins into which disease-associated mutations were engineered did not. In contrast, a cancer-associated mutation engineered into the rat CL1 GAIN domain and five cancer-associated mutations engineered into the human CL3 GAIN domain did not abrogate autoproteolysis, suggesting that the mechanism by which these mutations cause disease is distinct from that associated with ADPKD. Possible roles for the autocatalysis of the GAIN domain include aiding in receptor trafficking or locking the GAIN domain into a conformation that allows it to modulate receptor activity (see commentary by Tesmer).
D. Araç, A. A. Boucard, M. F. Bolliger, J. Nguyen, S. M. Soltis, T. C. Südhof, A. T. Brunger, A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis. EMBO J. 31, 1364–1378 (2012). [PubMed]
J. J. G. Tesmer, A GAIN in understanding autoproteolytic G protein-coupled receptors and polycystic kidney disease proteins. EMBO J. 31, 1334–1335 (2012). [PubMed]
Citation: A. M. VanHook, GAINing Autoproteolytic Activity. Sci. Signal. 5, ec93 (2012).
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