S49 cells endogenously express subtype 2 somatostatin receptors which couple to increase protein tyrosine phosphatase activity in membranes and down-regulate Raf-1 activity in situ.
S49 cells expressed type 2 somatostatin receptors (sstr2) by immunoblotting. Analysis by reverse transcription and polymerase chain reaction (RT-PCR) methodologies showed that S49 cells express predominantly sstr2A and sstr2B mRNAs; other subtypes were either not detected, in the case of sstr1, sstr3, sstr4, or variably detected, in the case of sstr5. No mutations were present in S49 cells at codon 12, 13, or 61 of the N-, K-, or H-ras genes. Nevertheless, randomly growing S49 cells contained Raf-1 activity by specific immune complex kinase assays. Treatment of S49 cells with somatostatin transiently inactivated the basal activity of Raf-1, but not that of B-Raf. Addition of somatostatin plus guanyl-5'-yl imidodiphosphate (GMPPNP) to S49 membranes stimulated PTPase activity. The concentration dependence for stimulation of PTPase activity correlated with high affinity binding of [125I-Tyr11]somatostatin-14. Both the effect of somatostatin to stimulate PTPase activity and to inactivate Raf-1 were abrogated by PTx. PTPase activity stimulated by somatostatin plus GMPPNP was recovered in a peak of high apparent M(r) (670,000) after solubilisation with Triton X-100 and Superose 6 chromatography. Furthermore, addition of activated, brain G alpha i/o subunits to fractions from control membranes stimulated PTPase activity in the high M(r) peak. Thus, S49 membranes contain a G-protein regulated PTPase (PTPase-G), and PTPase-G in these cells may reside in a high molecular weight complex.
Dent
P
P
Howard Hughes Medical Institute, Department of Medicine, University of Virginia, Charlottesville 22908, USA.
Wang
Y
Y
Gu
Y Z
YZ
Wood
S L
SL
Reardon
D B
DB
Mangues
R
R
Pellicer
A
A
Schonbrunn
A
A
Sturgill
T W
TW
D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D015536:Down-Regulation; D019204:GTP-Binding Proteins / Q000378:metabolism; D011905:Genes, ras; D051379:Mice; D009154:Mutation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D012333:RNA, Messenger; D017481:Receptors, Somatostatin / Q000096:biosynthesis* / Q000235:genetics; D013004:Somatostatin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D017481:Receptors, Somatostatin; D013004:Somatostatin; C093733:somatostatin receptor 2; D019908:Proto-Oncogene Proteins c-raf; D017027:Protein Tyrosine Phosphatases; D019204:GTP-Binding Proteins