Cloning and characterization of the annexin II receptor on human marrow stromal cells.
Annexin II is a heterotetramer, consisting of two 11-kDa (p11) and two 36-kDa (p36) subunits, that is produced by osteoclasts and stimulates osteoclast formation. However, its receptor is unknown. We showed that annexin II binds to normal primary human marrow stromal cells and the Paget's marrow-derived PSV10 stromal cell line to induce osteoclast formation. 125I-Labeled annexin II binding assays with PSV10 cells demonstrated that there was a single class of annexin II receptors with a Kd of 5.79 nm and Bmax of 2.13 x 10(5) receptors/cell. Annexin III or annexin V did not bind this receptor. Using 125I-labeled annexin II binding to screen NIH3T3 transfected with a human marrow cDNA expression library, we identified a putative annexin II receptor clone, which encoded a novel 26-kDa type I membrane receptor protein when expressed in HEK 293 cells. HEK 293 cells transformed with the cloned annexin II receptor cDNA showed a similar binding affinity to annexin II as that observed in PSV10 cells. Chemical cross-linking experiments with biotinylated annexin II and intact PSV10 cells identified a 55-kDa band on Western blot analysis that reacted with both an anti-p11 antibody and streptavidin but not anti-p36 antibody. A rabbit polyclonal antibody raised against the putative recombinant annexin II receptor also recognized the same 26-kDa protein band detected in PSV10 cells. Importantly, the annexin II receptor antibody dose-dependently blocked the stimulatory effects of annexin II on human osteoclast formation, demonstrating that the receptor mediates the effects of annexin II on osteoclast formation.
Lu
Ganwei
G
Medicine-Hematology/Oncology, University of Pittsburgh, Pittsburgh, Pennsylvania 15240, USA.
Maeda
Hidefumi
H
Reddy
Sakamuri V
SV
Kurihara
Noriyoshi
N
Leach
Robin
R
Anderson
Judith L
JL
Roodman
G David
GD
D000818:Animals; D017306:Annexin A2 / Q000737:chemistry*; D001854:Bone Marrow Cells / Q000378:metabolism*; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010010:Osteoclasts / Q000378:metabolism; D018000:Receptors, Peptide / Q000737:chemistry* / Q000235:genetics*; D017154:Stromal Cells / Q000378:metabolism*
D017306:Annexin A2; D003432:Cross-Linking Reagents; D018000:Receptors, Peptide; C514873:annexin II receptor, human