Catecholamine hormone receptor differences identified on 3T3 and simian virus-transformed 3T3 cells.
Identification and characterization of hormone receptors on the cell surface is an effective tool for studying the plasma membrane. Using the direct binding of a radiolabeled antagonist, (-)[3H]alprenolol, to crude membrane preparations, and a physiological response (cellular cyclic AMP levels), I demonstrated a catecholamine (beta-adrenergic) hormone receptor site coupled to a catecholamine responsive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] on 3T3 and simian virus 40 (SV40)-transformed 3T3 cells. At a concentration of 1 muM, epinephrine and isoproterenol elevate cellular cyclic AMP levels 8- and 12-fold, respectively, in both cell lines. Norepinephrine was also a potent agonist on 3T3 cells (8-fold stimulation), but SV3T3 cells showed a lesser (2-fold) response to this hormone. The specificity of the physiological response (as well as the direct binding studies using the alprenolol radiolabel) is indicated by the increased effectiveness of (-) compared to (+) stereoisomers, rapid and reversible kinetics (steady state within 2 min), high affinity (Kd approximately 30 nM) and saturability (indicating a finite number of hormone receptors). These hormone receptor studies indicate the 3T3 cells have a beta1 adrenergic receptor while the SV3T3 cells have a receptor with beta2 qualities. In addition, the number of beta-adrenergic hormone receptors appear to be increased in the normal 3T3 cells by approximately 2-fold over the SV3T3 cells (300 versus versus 120 femtomol/mg of protein).
Catecholamine hormone receptor differences identified on 3T3 and simian virus-transformed 3T3 cells. Identification and characterization of hormone receptors on the cell surface is an effective tool for studying the plasma membrane. Using the direct binding of a radiolabeled antagonist, (-)[3H]alprenolol, to crude membrane preparations, and a physiological response (cellular cyclic AMP levels), I demonstrated a catecholamine (beta-adrenergic) hormone receptor site coupled to a catecholamine responsive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] on 3T3 and simian virus 40 (SV40)-transformed 3T3 cells. At a concentration of 1 muM, epinephrine and isoproterenol elevate cellular cyclic AMP levels 8- and 12-fold, respectively, in both cell lines. Norepinephrine was also a potent agonist on 3T3 cells (8-fold stimulation), but SV3T3 cells showed a lesser (2-fold) response to this hormone. The specificity of the physiological response (as well as the direct binding studies using the alprenolol radiolabel) is indicated by the increased effectiveness of (-) compared to (+) stereoisomers, rapid and reversible kinetics (steady state within 2 min), high affinity (Kd approximately 30 nM) and saturability (indicating a finite number of hormone receptors). These hormone receptor studies indicate the 3T3 cells have a beta1 adrenergic receptor while the SV3T3 cells have a receptor with beta2 qualities. In addition, the number of beta-adrenergic hormone receptors appear to be increased in the normal 3T3 cells by approximately 2-fold over the SV3T3 cells (300 versus versus 120 femtomol/mg of protein).