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1.
J Biol Chem ; 274(35): 25093-8, 1999 Aug 27.
Article in English | MEDLINE | ID: mdl-10455189

ABSTRACT

HEF1 is a recently described p130(Cas)-like docking protein that contains one SH3 domain and multiple SH2 binding motifs. In B cells, HEF1 is phosphorylated by a cytoskeleton-dependent mechanism that is triggered by integrin ligation. However, the induction of HEF1 phosphorylation by G protein-coupled receptors has not been reported. We found that HEF1, but not p130(Cas), is tyrosine-phosphorylated following stimulation of the rabbit C1a calcitonin receptor stably expressed in HEK-293 cells. The calcitonin-induced tyrosine phosphorylation of HEF1 increased in a time- and dose-dependent manner. Dibutyryl cAMP and forskolin had little or no effect on HEF1 phosphorylation, and the protein kinase A inhibitor H89 failed to detectably inhibit the response to calcitonin, indicating that the G(s)/cAMP/protein kinase A pathway does not mediate the calcitonin effect. Pertussis toxin, which selectively blocks G(i/o) signaling, also had no effect. Increasing cytosolic Ca(2+) with ionomycin stimulated HEF1 phosphorylation and preventing any calcitonin-induced change in cytosolic calcium by a combination of BAPTA and extracellular EGTA completely blocked the calcitonin-induced tyrosine phosphorylation of HEF1. Phorbol 12-myristate 13-acetate also induced HEF1 tyrosine phosphorylation, and the protein kinase C inhibitor calphostin C completely inhibited both calcitonin- and phorbol 12-myristate 13-acetate-stimulated HEF1 phosphorylation. Calcitonin also induced the tyrosine phosphorylation of paxillin and focal adhesion kinase, and the association of these two proteins with HEF1. Pretreatment with cytochalasin D, which disrupts actin microfilaments, prevented the calcitonin-induced HEF1 and paxillin phosphorylation. In conclusion, the calcitonin-stimulated tyrosine phosphorylation of HEF1 is mediated by calcium- and protein kinase C-dependent mechanisms and requires the integrity of the actin cytoskeleton.


Subject(s)
Calcitonin/pharmacology , Cytoskeleton/metabolism , GTP-Binding Proteins/metabolism , Phosphoproteins/metabolism , Proteins , Receptors, Calcitonin/metabolism , Adaptor Proteins, Signal Transducing , Calcium/pharmacology , Cell Adhesion Molecules/metabolism , Cell Line , Crk-Associated Substrate Protein , Cytochalasin D/pharmacology , Cytoskeletal Proteins/metabolism , Enzyme Activation , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Paxillin , Phosphorylation , Phosphotyrosine/metabolism , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/metabolism , Retinoblastoma-Like Protein p130 , Signal Transduction
2.
J Bone Miner Res ; 14(8): 1425-31, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10457276

ABSTRACT

In this study we characterized the biological activity of the recently identified salmon calcitonin (sCT) IV, in order to evaluate its potential therapeutic value. In the rat bioassay, sCT IV exhibited a 30% higher hypocalcemic activity than sCT I. The capacity of the molecule to inhibit bone resorption was assessed in vitro by the bone resorbing assay and the pit assay. An inhibitory effect, similar to that of sCT I, was observed in both assays. The interaction of sCT IV with the rabbit CT receptor was also studied. The affinity of sCT IV for the receptor was similar to that of sCT I, as was the potency for stimulating cAMP production. The antigenicity of the two molecules was not identical. Thus, this new CT could represent a useful novel therapeutic agent for the treatment of bone disorders.


Subject(s)
Calcitonin/physiology , Protein Isoforms/physiology , Animals , Antigens/immunology , Biological Assay , Bone Resorption/physiopathology , Calcitonin/immunology , Cross Reactions , Cyclic AMP/biosynthesis , Female , Logistic Models , Male , Pregnancy , Rabbits , Rats , Rats, Sprague-Dawley , Rats, Wistar , Receptors, Calcitonin/physiology
3.
Eur J Biochem ; 262(1): 95-101, 1999 May.
Article in English | MEDLINE | ID: mdl-10231369

ABSTRACT

The calcitonin receptor is known to couple to Gs and Gq, activating adenylyl cyclase and phospholipase C, respectively. The observation of pertussis-toxin-sensitive responses to calcitonin suggests that the receptor is capable of coupling to Gi/o as well. However, the calcitonin-dependent activation of adenylyl cyclase in HEK-293 cells that stably express the cloned rabbit calcitonin receptor, as in many other cells that express calcitonin receptors, shows little pertussis toxin sensitivity. Calcitonin treatment of these cells stimulates protein kinase C, which is reported to antagonize the receptor-dependent activation of Gi. The possibility that protein kinase C could be antagonizing Galphai-adenylyl cyclase coupling was tested by examining the effects of protein kinase C inhibitors (chelerythrine chloride and sphingosine) or of chronic treatment with phorbol ester to deplete protein kinase C. All three treatments led to a reduction of calcitonin-induced adenylyl cyclase activity that was reversed by pertussis toxin. Inhibiting or depleting protein kinase C had no effect on the activation of adenylyl cyclase by cholera toxin, indicating that Gs and adenylyl cyclase were not affected by these treatments. Calcitonin treatment of HEK-293 cells, that stably express a myc-tagged rabbit calcitonin receptor, induced the formation of complexes of the receptor and Galphai subunits, confirming that the calcitonin receptor interacts with Gi. Thus, the calcitonin receptor can couple to Gi, but the inhibition of adenylyl cyclase by Galphai is negatively regulated by protein kinase C.


Subject(s)
Adenylate Cyclase Toxin , Adenylyl Cyclases/metabolism , Pertussis Toxin , Protein Kinase C/metabolism , Receptors, Calcitonin/metabolism , Virulence Factors, Bordetella/pharmacology , Amino Acid Sequence , Animals , Calcitonin/antagonists & inhibitors , Cell Line , Enzyme Activation , Humans , Molecular Sequence Data , Protein Binding , Rabbits
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