Parathyroid hormone-related protein. Evidence for secretion of a novel mid-region fragment by three different cell types.
The cDNA-predicted amino acid sequence of parathyroid hormone-related protein (PTHrP) contains multiple basic amino acid motifs, suggesting that PTHrP undergoes extensive post-translational processing prior to secretion. The secretory forms of the peptide are currently unknown. To identify these secretory forms, medium was harvested from three cell types: human renal carcinoma (SKRC-1) cells, human keratinocytes, and rat insulinoma cells stably transfected with the cDNA for PTHrP(1-141) (RIN-141 cells). Amino-terminal species were immunopurified using an anti-PTHrP(1-36) column, and mid-region species using an anti-PTHrP(37-74) column. PTHrP peptides in medium and in cell extracts were further resolved by reverse phase high performance liquid chromatography (RP-HPLC) and identified using region-specific immunoassays. SKRC-1 and RIN-141 cells secreted three distinct amino-terminal species and a novel, non-amino-terminal, mid-region fragment. Sequence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the RIN-141 cell mid-region fragment begins at amino acid 38 of the cDNA-predicted sequence and is approximately 70 amino acids in length. Comparison of RP-HPLC elution patterns suggests that SKRC-1 cells and keratinocytes secrete a similar or identical mid-region fragment. Immunofluorescence studies revealed a Golgi pattern for the amino-terminal species and a secretory granule pattern for the mid-region fragment. These studies indicate that 1) multiple PTHrP species are secreted, including a novel mid-region fragment; 2) Arg37 serves as a cleavage site in at least three cell types; 3) PTHrP(1-36) is likely to be an authentic secretory form of PTHrP; and 4) the mid-region fragment appears to be packaged into secretory granules. The marked interspecies conservation of this mid-region PTHrP suggests that it will have important biological functions.
Soifer
N E
NE
Division of Endocrinology, West Haven Veterans Affairs Medical Center, Connecticut 06516.
Dee
K E
KE
Insogna
K L
KL
Burtis
W J
WJ
Matovcik
L M
LM
Wu
T L
TL
Milstone
L M
LM
Broadus
A E
AE
Philbrick
W M
WM
Stewart
A F
AF
D000595:Amino Acid Sequence; D000818:Animals; D002292:Carcinoma, Renal Cell; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D007680:Kidney Neoplasms; D007700:Kinetics; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000235:genetics; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000302:isolation & purification; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis / Q000235:genetics*; D014162:Transfection
C470121:PTHLH protein, human; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D011506:Proteins