Abstract
Coupling between D-1 dopamine receptors and G proteins in cell lines expressing human D-1 receptors and different G proteins was examined. Pertussis toxin (PTX) treatment of rat pituitary GH4C1 cells significantly reduced, but did not abolish, agonist high affinity binding sites of the D-1 dopamine receptor; in SK-N-MC neuroblastoma cells, PTX failed to have any effect on D-1 high affinity sites. Cholera toxin (CTX) treatment of GH4C1 cells reduced but did not abolish the high affinity sites of D-1 receptors, while in SK-N-MC cells, treatment with CTX abolished all the high affinity sites. Western blot analyses with specific antisera indicated that Gs alpha, Gi1 alpha, Gi3 alpha, and Gq alpha were expressed in both cell lines, while Gi2 alpha and G(o) alpha were expressed in GH4C1 but not SK-N-MC cells. Antisera NEI-805 (anti-Gs alpha) and 9072 (anti-G(o) alpha) immunoprecipitated 24 +/- 4.3 and 34.4 +/- 6.9%, respectively, of G protein-associated D-1 dopamine receptors. Antisera 3646 (anti-Gi1 alpha), 1521 (anti-Gi2 alpha), 1518 (anti-Gi3 alpha), and 0941 (anti-Gq alpha) failed to coimmunoprecipitate appreciable levels of soluble receptors. These data indicate that D-1 dopamine receptors are coupled to both Gs alpha and G(o) alpha but not to Gq alpha.
Highlights
Pertussis toxin (PTX) treatment of rat pituitary GH4C1 cells significantly reduced, but did not abolish, agonist high affinity binding sites of the D·l dopamine receptor; in SK-N-MC neuroblastoma cells, PTX failed to have any effect on D-l high affinity sites
Western blot analyses with specific antisera indicated that Gsa, Gil a, GiSa, and Gqa were expressed in both cell lines, while Gi2a and Goa were expressed in GH4C1 but not SK-N-MC cells
Immunodetection of the G Proteins Expressed in GH4C1 and SK-N-MC Cells-Since different cells express different G proteins, we examined the expression of PTX-sensitive G proteins in GH 4C I and SK-N-MC cells, which could account for the inability of D-1 sites to display PTX-sensitive couplings in SK-N-MC cells
Summary
Materials-All drugs used in this study were obtained from Research Biochemicals Inc. (Natick, MA); the D-1-selective radioligand, 8-iodo2,3,4,5-tetrahydro-3-methyl-5-phenyl-lH-3-benzazepine-7-01, 1Z5I-SCH 23982 (Sidhu, 1990), was purchased from DuPont NEN. Membranes from GH 4C1 cells were suspended in solubilization buffer (50 mM Tris-HCI, pH 7.4,1 MNaCI, 5 mx KCI, 2 mM csci; 1 mM MgCl z, 250 mM sucrose, 1 mM DTT, 1 mM EDTA, 5 J.Lg each ofleupeptin and pepstatin, and 1 mM phenylmethylsulfonyl fluoride) at a protein concentration of 1-1.5 mg/ml. The samples were incubated for an additional 90 min and centrifuged at 16,000 x g for 5 min in a microfuge; the supernatant was removed and saved as the "supernatant fraction." The pellets were washed once in buffer A containing protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride and 5 J.Lg each of leupeptin and pepstatin) and resuspended in 200-400 J.LI of binding assay buffer. D-1 dopamine receptors in both the pellet fraction and the supernatant fraction (after reconstitution into phospholipid vesicles) were assayed using the same binding assay procedures and buffers, described above for membranebound receptors. All values represent means ± S.D. from separate, independent experiments where n equals the number of experiments
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
