Effects of proteolytic enzymes and monovalent ions demonstrate protease-sensitive and protease-insensitive stereospecific binding sites on dopaminergic receptors in rat striatum.
Rat striatal membranes from different subcellular fractions were treated with various proteolytic and other enzymes and the binding of a dopamine agonist ([3H]NPA) and of an antagonist ([3H]haloperidol) was assayed in several conditions. In membranes of striatal microsomal and mitochondrial fractions, stereospecific binding of both [3H]NPA and [3H]haloperidol assayed in a monovalent ion-poor buffer was potently and rapidly inhibited by trypsin and certain related proteases. The enzymes did not affect the binding  of the ligands when assayed in a buffer containing monovalent ions (greater than or equal to 40 mM NaCl or KCl or a physiological mixture of electrolytes). The inhibition, seen in the monovalent ion-poor buffer, was dependent on the enzyme concentration. The endoproteases (trypsin, alpha-chymotrypsin, papain, ficin) showed nanomolar IC50-values for inhibition of both [3H]NPA and [3H]haloperidol binding. The inhibition occurred very rapidly at 0 degree and was different from the slow proteolytic inactivation seen by prolonged incubation at 37 degrees. It was demonstrated that monovalent ions did not themselves interfere with the interaction between the proteases and the membranes. The observations provide evidence for two different types of stereospecific dopaminergic binding sites which are differentially exposed for ligand binding depending on the concentration of monovalent ions. There sites are protease-sensitive sites, labelled in monovalent ion-poor media and protease-insensitive sites, labelled in media with higher concentrations of monovalent ions. Both types of binding sites bind dopamine agonists and dopamine antagonists with high affinity, but some differences were noted in the binding properties and the drug binding selectivity of the sites. It is argued that both sites form part of the same dopamine receptor macromolecular complex. The findings corroborate the hypothesis that dopamine receptors are composed of different sub-unit binding sites, but these are not distinct agonist and antagonist specific sites. The mechanism by which the protease-sensitive sites are rapidly inactivated by particular proteases, is probably a complexation between the enzymes and certain essential peptide moieties of the receptor sites involved.
D000818:Animals; D001058:Apomorphine / Q000031:analogs & derivatives / Q000378:metabolism; D002414:Cations, Monovalent; D003342:Corpus Striatum / Q000378:metabolism*; D005260:Female; D006220:Haloperidol / Q000378:metabolism; D066298:In Vitro Techniques; D008566:Membranes / Q000378:metabolism; D010447:Peptide Hydrolases / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D011954:Receptors, Dopamine / Q000378:metabolism*; D013237:Stereoisomerism
D002414:Cations, Monovalent; D011954:Receptors, Dopamine; C009074:N-n-propylnorapomorphine; D010447:Peptide Hydrolases; D006220:Haloperidol; D001058:Apomorphine