Catalytic activation of guanylate cyclase/atrial natriuretic factor receptor by combined effects of ANF and GTP gamma S in plasma membranes of Leydig tumor cells: involvement of G-proteins.
Guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) exhibited a modulatory role in the catalytic activation of guanylate cyclase-A/atrial natriuretic factor receptor (GC-A/ANF-R) in the plasma membrane preparations of murine Leydig tumor (MA-10) cells. Both atrial natriuretic factor (ANF) and GTP gamma S synergistically stimulated the guanylate cyclase (GC) activity of GC-A/ANF-R in a dose- and time-related manner. Other nucleotides and their analogs such as ATP, adenosine 5'-(gamma-thio)triphosphate, adenosine 5'-(beta,gamma-imino)triphosphate, GDP, and guanosine 5'-(2-O-thiodiphosphate) (100 microM each) did not show any discernible effect on GC catalytic activity of GC-A/ANF-R. A significant stimulation of GC activity was observed in the presence of mastoparan, AlF4-, and benzalkonium chloride. The saturation binding assay of [35S]GTP gamma S showed the dissociation constant (Kd) of 2.3 x 10(-9) M and the binding capacity (Bmax) of 76 pmol/mg protein in the plasma membrane preparations of MA-10 cells. ANF increased the [35S]GTP gamma S-binding capacity, however, without affecting its affinity constant. Pretreatment of plasma membranes with antibodies against Gs alpha subunit attenuates the GTP gamma S-stimulated GC activity, whereas antibodies against Gi alpha subunit enhanced the stimulatory effect of GTP gamma S on GC catalytic activity of GC-A/ANF-R. However, the antibodies against Go alpha subunit did not show any effect on GC activity. These results provide the evidence that both Gs and Gi subunits of G-proteins seem to be involved in the regulation of GC catalytic activity of GC-A/ANF-R in the plasma membranes of MA-10 cells.
Department of Biochemistry and Molecular Biology, Medical College of Georgia, School of Medicine, Augusta 30912-2100.
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ML
Khurana
K N
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Pandey
D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D001548:Benzalkonium Compounds / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006162:Guanylate Cyclase / Q000187:drug effects* / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D007984:Leydig Cell Tumor / Q000378:metabolism; D051379:Mice; D009711:Nucleotides / Q000494:pharmacology; D010455:Peptides; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000187:drug effects* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000187:drug effects* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014862:Wasp Venoms / Q000494:pharmacology
D017607:Aluminum Compounds; D001548:Benzalkonium Compounds; D036341:Intercellular Signaling Peptides and Proteins; D009711:Nucleotides; D010455:Peptides; D011956:Receptors, Cell Surface; D014862:Wasp Venoms; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C037500:mastoparan; D009320:Atrial Natriuretic Factor; D019204:GTP-Binding Proteins; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A; D005459:Fluorides; C032311:aluminum fluoride