Heat shock protein-70 expressed on the surface of cancer cells binds parathyroid hormone-related protein in vitro.
Recent studies have shown that the functions of PTH-related protein (PTHrP) and its derived peptides cannot be attributed solely to PTH/PTHrP receptor binding. The present study focused on the identification of other proteins that might bind PTHrP at the cell surface. Using affinity chromatography, we applied extracts of cell-surface biotinylated proteins from cancer and normal cell lines over Sepharose beads coupled with different PTHrP-derived peptides. Elution with the corresponding free peptide revealed a major protein of about 70 kDa that was present in all of the PTHrP peptide eluates from cancer cell extracts but not from normal breast cell extracts. Mass spectroscopy analysis and immunoblotting identified this PTHrP-binding protein as heat shock protein-70 (HSP70). Using a recently published algorithm that predicts HSP70 binding sites within proteins, we found that all four PTHrP peptides used in these studies contain amino acid motifs with high probabilities for HSP70 binding in vivo. Cell culture studies in the presence of a polyclonal anti-HSP70 antibody demonstrated increased PTHrP secretion, decreased total cellular protein, and differentially regulated proliferation. Taken together, these studies demonstrate a novel and biologically relevant interaction between cell surface-expressed HSP70 and PTHrP in cancer.
Grzesiak
John J
JJ
Department of Surgery, University of California, and Veterans Affairs San Diego Healthcare System, 3350 La Jolla Village Drive, San Diego, California 92161, USA.
Smith
Kathy C
KC
Chalberg
Cheryl
C
Truong
Caitlyn
C
Burton
Douglas W
DW
Deftos
Leonard J
LJ
Bouvet
Michael
M
D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D019921:Biotinylation; D001859:Bone Neoplasms; D002455:Cell Division; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000502:physiology; D002846:Chromatography, Affinity; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D012516:Osteosarcoma; D044162:Parathyroid Hormone-Related Protein / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011471:Prostatic Neoplasms
D018840:HSP70 Heat-Shock Proteins; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments