Role of cysteine residues of rat A2a adenosine receptors in agonist binding.
In the present study, we investigated the role of disulfide bridges and sulfhydryl groups in A2a adenosine receptor binding of the agonist 2-p-(2-carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoadenosi ne (CGS 21680). To evaluate the presence of essential disulfide bridges, rat striatal membranes were incubated with [3H]CGS 21680 in the presence of dithiothreitol and binding of the agonist to membranes was measured. The amount of [3H]CGS 21680 which specifically bound, decreased progressively upon pretreatment of membranes with increasing concentrations of dithiothreitol. Pretreatment of rat striatal membranes with 12.5 mM dithiothreitol for 15 min at 25 degrees C resulted in a 2-fold decrease of A2a adenosine receptor affinity for [3H]CGS 21680, and a reduction in the maximal number of binding sites. The presence of agonist or antagonist ligands protected the A2a adenosine receptor sites from the effect of dithiothreitol. We also examined the susceptibility of A2a adenosine receptors to inactivation by the sulfhydryl alkylating reagent, N-ethylmaleimide. When rat striatal membranes were pretreated with N-ethylmaleimide for 30 minutes at 37 degrees C, a decrease in specific [3H]CGS 21680 binding was observed. Pretreatment of membranes with 1 mM N-ethylmaleimide also resulted in a 2-fold reduction of A2a adenosine receptor affinity for [3H]CGS 21680, as well as a slight decrease in the maximal number of binding sites. Neither agonist nor antagonist ligands were effective in protecting the receptor sites from inactivation by N-ethylmaleimide. In contrast, addition of 100 microM guanosine-5'-O-(3-thiotriphosphate) or 5'-guanylylimidodiphosphate were both effective in protecting the receptor sites from inactivation by N-ethylmaleimide. This protective effect was significant but not complete. Our data suggest that disulfide bridges play a role in the structural integrity of the A2a adenosine receptor, furthermore, reduced sulfhydryl groups appear to be important but we do not yet know if they are on the receptor or on the Gs alpha subunit.
Mazzoni
M R
MR
Istituto Policattedra di Discipline Biologiche, University of Pisa, Italy. mariarm@farm.unipi.it
Giusti
L
L
Rossi
E
E
Taddei
S
S
Lucacchini
A
A
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019830:Adenosine-5'-(N-ethylcarboxamide); D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003342:Corpus Striatum; D003545:Cysteine / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D008297:Male; D010627:Phenethylamines / Q000378:metabolism*; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D010627:Phenethylamines; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D018047:Receptors, Purinergic P1; D013439:Sulfhydryl Reagents; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D006165:Guanylyl Imidodiphosphate; D019830:Adenosine-5'-(N-ethylcarboxamide); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000241:Adenosine; D003545:Cysteine; D005033:Ethylmaleimide; D004229:Dithiothreitol