J. Biochem, 1989, Vol. 106, No. 1 17-22
© 1989 Japanese Biochemical Society
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Deglycosylation and Proteolysis of Photolabeled D2 Dopamine Receptors of the Porcine Anterior Pituitary1
*Departments of Pharmacology, University of Toronto Toronto, Ontario, M5S 1A8 Canada
**Departments of Psychiatry, University of Toronto Toronto, Ontario, M5S 1A8 Canada
2To whom correspondence should be addressed: Dept. of Pharmacology, University of Toronto, Toronto, Ontario, M5S 1A8 Canada. Abbreviations: [125I]N3NAPS, [125I]N-aadophenethylspiperone; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PNGase-F, glycopeptide-iV-glycosidase; BTC, butaclamol.
Dopamine D2 receptor binding subunits of the porcine anterior pituitary were visualized by autoradiography following photoaffinity labeling with [125I]N-azidophenethylspiperone and sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The ligand binding subunit comprising the pituitary D2 dopamine receptor migrated as two distinct bands of apparent Mr
150, 000 and 118, 000, substantially higher than neuronal D2 receptor subunits from porcine or canine brain. The glycoprotein nature of pituitary D2 receptor binding subunits was investigated by the use of exo- and endo-glycosidase treatments and peptide mapping experiments. Photoaffinity labeled polypeptides of the anterior pituitary were susceptible to both neuraminidase and 
-mannosidase digestion as indexed by their increased electrophoretic mobility on sodium dodecyl-sulfate polyacrylamide gels, and suggests the presence of both complex type and terminal mannose carbohydrate residues. Moreover, the additive effects of sequential treatment with these enzymes suggests that both types of carbohydrate chains are present on each receptor peptide. N-linked deglycosylation of pituitary D2 photolabeled receptors with glycopeptidase-F produced a further increase in the mobility of the labeled protein to apparent Mr 44, 000, similar to that of de-glycosylated D2 binding subunits of porcine and canine brain. Peptide mapping experiments following limited proteolysis with Staphylococcus aureus V8 proteinase and papain demonstrated that deglycosylated D2 dopamine receptors (Mr = 44, 000), in different tissues and species, were homologous. Taken together, these data suggest that despite the differences in the overall molecular weight and tissue specific glycosylation pattern of pituitary D2 dopamine receptors, the primary structure of mammalian D2 receptors appears to be conserved.
1K.R.J. is a recipient of an OMHF studentship, and H.B.N. is a Career Scientist of the Ontario Ministry of Health HRPDP. Portions of this work were supported by the MRC of Canada, NARSAD and the Canadian Friends of Schizophrenics.