Pretreatment of human platelet membranes with trypsin abolishes GTP but not Na+ effects on alpha 2-adrenoreceptor-agonist interactions.
The affinity of many types of membrane-bound receptors coupled negatively to adenylate cyclase is regulated by divalent and monovalent cations and by guanine nucleotides (GTP). We used alpha 2-adrenoreceptors of human platelets as a model system to find out the effect of limited proteolysis with trypsin on the regulation of the alpha 2-adrenoreceptor-agonist interactions by GTP and Na+. We found that partial proteolysis of the membranes with trypsin for 3 min at 35 degrees C reduced specific [3H]yohimbine binding to platelet membranes to 40-50% of control. The following characteristics of the receptors remaining after proteolysis were similar to those of untreated membranes: affinity for the agonist and antagonists, stereospecificity, and kinetic properties. Trypsin also did not modify the ability of the receptor's change from a high to low affinity state in the presence of Na+. These findings suggested that the capability of the receptors to recognize the ligand and their ability to undergo a conformational change in the presence of the agonist were retained despite a reduction in the total number of receptors by trypsin. However, the modulation of the receptor--agonist interactions by GTP or Mg2+ was lost in the trypsin-pretreated membranes, while the modulation by Na+ remained intact. It is suggested that the loss of GTP or Mg2+ effects on receptor--ligand interactions produced by trypsin may be due to trypsin-induced disruption of subunits (alpha i, beta gamma) interactions of Gi protein.
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D004837:Epinephrine / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011869:Radioligand Assay; D011942:Receptors, Adrenergic, alpha / Q000502:physiology*; D012964:Sodium / Q000502:physiology*; D014357:Trypsin / Q000494:pharmacology; D015016:Yohimbine / Q000378:metabolism / Q000494:pharmacology
D000316:Adrenergic alpha-Agonists; D011942:Receptors, Adrenergic, alpha; D015016:Yohimbine; D006160:Guanosine Triphosphate; D012964:Sodium; D014357:Trypsin; D008274:Magnesium; D004837:Epinephrine