Cloning and characterization of a novel G protein beta-subunit of pearl oyster (Pinctada fucata), and its interaction sites with calmodulin.
A cDNA clone encoding a novel G protein beta subunit of beta1 subclass, pfGbeta1 was isolated from the pearl oyster (Pinctada fucata). The deduced amino acid sequence of pfGbeta1 (341 amino acids) shares high homology to northern European squid (Loligo pealei) and great pond snail (Lymnaea stagnalis) pfGbeta1, while it has diverged from bovine (Bos taurus) and human. The well-conserved amino acid domains in G protein beta subunit, seven WD repeats, were founded in the deduced amino acid sequence. Alignment analysis showed that the beginning amino acid residues in variable fragment of the seventh WD motif are different from any other Gbeta. The prediction of 3D structure of pfGbeta showed that pfGbeta belongs to beta-propeller family proteins whose members contain 4-8 antiparallel beta-sheets resembling the blades of a propeller. In situ hybridization and Northern blotting analysis revealed that the pfGbeta mRNA hybridization signals were widely expressed in various tissues except muscle, with abundantly in epithelia of gill, gonad and outer fold of mantle. We also investigated the interactions between Gbetagamma and calmodulin (CaM), and specific amino acid residues that may be critical for the binding of Gbetagamma to CaM were also identified. Furthermore, the functional studies of the interaction showed that the binding of CaM and Gbetagamma increases the alkaline phosphatase (ALP) activity, an indicator for mineralization in MC3T3-E1 cells. The ALP activity of the mutants of pfGbetagamma that impaired the interactions of Gbetagamma with CaM is higher than the Control group; however, it is lower than the WTC group. Together, these results suggest that the Gbetagamma might interact with CaM and point to the important physiological function in modulating cellular functions.
Chen
Lei
L
Institute of Marine Biotechnology, Department of Biological Science and Biotechnology, Tsinghua University, Beijing, 100084, PR China.
Xie
Liping
L
Xiong
Xunhao
X
Dai
Yiping
Y
Fan
Weimin
W
Zhang
Rongqing
R
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D044387:GTP-Binding Protein beta Subunits / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D010802:Phylogeny; D049911:Pinctada / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D016415:Sequence Alignment
D002147:Calmodulin; D018076:DNA, Complementary; D044387:GTP-Binding Protein beta Subunits; D012333:RNA, Messenger; D000469:Alkaline Phosphatase