Co-purification of a protein tyrosine phosphatase with activated somatostatin receptors from rat pancreatic acinar membranes.
We have previously shown that somatostatin promotes the stimulation of a membrane tyrosine phosphatase activity in pancreatic cells. To gain insight into the mechanism of somatostatin action, we purified somatostatin-receptor complexes from somatostatin 28-prelabelled rat pancreatic plasma membranes by immunoaffinity chromatography using immobilized antibodies raised against the N-terminal part of somatostatin 28, somatostatin 28 (1-14), which is not involved in receptor-binding-site recognition. After SDS gel electrophoresis a band with a molecular mass of 87 kDa was identified in the affinity-purified material as the somatostatin receptor. The 87 kDa protein was not observed when the membrane receptors were solubilized in a free unoccupied or somatostatin 14-occupied form, or when nonimmune serum replaced the anti-[somatostatin 28 (1-14)] anti-serum. Somatostatin 14 inhibited the appearance of the 87 kDa protein in the same range of concentrations that inhibit radioligand binding on pancreatic membranes. After somatostatin 28 treatment of membranes, purified somatostatin receptor preparations exhibited an elevated tyrosine phosphatase activity that dephosphorylated phosphorylated epidermal growth factor receptor and poly(Glu,Tyr). This activity was related to the presence of somatostatin receptors in purified material. It was increased by dithiothreitol and inhibited by orthovanadate. In purified material containing somatostatin receptors, anti-[Src homology 2 domains (SH2)]-containing tyrosine phosphatase SHPTP1 polyclonal antibodies identified a protein of 66 kDa which was not detected in the absence of somatostatin receptor. Furthermore, the anti-SHPTP1 antibodies immunoprecipitated specific somatostatin receptors from somatostatin-prelabelled pancreatic membranes and from untreated membranes. These results indicate that a 66 kDa tyrosine phosphatase related to SHPTP1 co-purifies with the pancreatic somatostatin receptors, and suggest that this protein is associated with somatostatin receptors at the membrane level.
Zeggari
M
M
INSERM U151, CHU Rangueil, Toulouse, France.
Esteve
J P
JP
Rauly
I
I
Cambillau
C
C
Mazarguil
H
H
Dufresne
M
M
Pradayrol
L
L
Chayvialle
J A
JA
Vaysse
N
N
Susini
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology* / Q000648:ultrastructure; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011498:Protein Precursors; D017027:Protein Tyrosine Phosphatases / Q000302:isolation & purification* / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D017481:Receptors, Somatostatin / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D013004:Somatostatin / Q000737:chemistry / Q000276:immunology; D053561:Somatostatin-28; D014638:Vanadates / Q000494:pharmacology
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011498:Protein Precursors; D017481:Receptors, Somatostatin; C027606:L-glutamic acid-L-tyrosine copolymer; D014638:Vanadates; D013004:Somatostatin; D053561:Somatostatin-28; D066246:ErbB Receptors; D017027:Protein Tyrosine Phosphatases; D004229:Dithiothreitol