Palmitoylation and its effect on the GTPase-activating activity and conformation of RGS2.
Regulator of G protein signaling (RGS) proteins act as negative regulators of G protein coupled signaling by accelerating the GTPase activity of the G proteins alpha subunits. Reversible palmitoylation, a common post-translational modification for various components of the G protein-coupled signaling pathway, plays an important role in the modulation of protein activity. RGS2 appears to act selectively to increase the GTPase activity of Gqalpha when single turnover assays are preformed in solution. However, less attention has been paid to the effects of palmitoylation of RGS2 on its conformation and GTPase-activating activity. Studies of palmitoylation on a series of RGS2 mutants in which alanine was substituted for cysteine revealed cysteine 106, 116 and 199 to be multiple putative palmitoylation sites in RGS2, the efficiency of palmitate incorporation being about 60% at each individual palmitoylation site. Palmitoylation of RGS2 inhibited the GTPase-activating activity toward a GTPase-deficient R183C mutant of Gqalpha in vitro, but mutation of cysteine 116 eliminated the inhibition of palmitoylation on GTPase-activating activity of RGS2. The effect of palmitoylation on conformation of RGS2 was examined by monitoring spectra of the intrinsic fluorescence and Circular Dichroism. The results suggested that GTPase-activating activity change of RGS2 might be related to conformational change of RGS2 upon palmitoylation. Taken together, these results provided clear and strong experimental evidence for palmitoylation sites in RGS2 as well as for effect of palmitoylation on the GTPase-activating activity and conformation of RGS2.
Ni
Jianqiang
J
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China. nijq@moon.ibp.ac.cn
Qu
Liang
L
Yang
Hui
H
Wang
Mengxi
M
Huang
Youguo
Y
D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D005453:Fluorescence; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000302:isolation & purification; D019308:Palmitic Acid / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D020710:RGS Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012996:Solutions; D014364:Tryptophan / Q000378:metabolism
D020690:GTPase-Activating Proteins; D050505:Mutant Proteins; D020710:RGS Proteins; D012996:Solutions; D019308:Palmitic Acid; D014364:Tryptophan; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D003545:Cysteine