Anti-mu opioid antiserum against the third external loop of the cloned mu-opioid receptor acts as a mu receptor neutral antagonist.
The region from the third external loop to the C terminus of MOR-1 appeared to be critical to the selective binding of MOR-1 ligands as DAMGO and morphine to MOR-1. To study the pharmacological properties of the third extracellular loop an antibody was raised in rabbits against the sequence 304-316 which is unique to MOR-1 and includes the third external loop; the anti-MOR-1 antibody was affinity purified against the immunogen sequence and characterized by [3H]DAMGO and Western blotting; [3H]DPDPE binding assay remained unchanged in the presence of the antibody. Anti-MOR-1 IgG was characterized as a neutral antagonist in Chinese hamster ovary (CHO) cells hyperexpressing constitutively active MOR-1s; in fact, anti-MOR-1 IgG completely reversed the inhibition induced by the MOR-1 agonist endomorphin1, endomorphin2, DAMGO and morphine on forskolin stimulated cyclic AMP (cAMP) accumulation and attenuated both the action of the selective MOR-1 agonist DAMGO to increase [35S]GTPgammaS binding and the action of the MOR-1 inverse agonist beta-chlornaltrexamine (CNA) to decrease [35S]GTPgammaS binding. Radioligand binding assay using membrane suspensions from CHO cells hyperexpressing MOR-1 revealed a significant decreased binding affinity and capacity of all the tested MOR-1 selective ligands after preincubation with anti-MOR-1 IgG. Therefore, the third extracellular loop of MOR-1 appeared to be a key element for the binding of MOR-1 ligands.
Department of Anatomical and Biomedical Sciences, University of Siena, Siena, Italy.
M
M
Guarna
A
A
Bartolini
C
C
Ghelardini
N
N
Galeotti
L
L
Bracci
G B
GB
Stefano
E
E
Bianchi
D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects / Q000276:immunology; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000276:immunology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008024:Ligands; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D011817:Rabbits; D043562:Receptors, G-Protein-Coupled / Q000276:immunology* / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D000906:Antibodies; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP