Nourdine Amlaiky

Identification of the D2‐‐Dopamine Receptor Binding Subunit in Several Mammalian Tissues and Species by Photoaffinity Labeling

Photoaffinity labeling of the D2‐‐dopamine receptor in plasma membrane preparations of various tissues from several mammalian species was performed using the recently developed D2‐‐dopaminergic antagonist probe [125I]N‐(p‐azidophenethyl)spiperone ([125I]N3‐‐NAPS). In tissues containing D2‐‐receptors such as the corpus striatum from rat, dog, calf, hamster, guinea pig, and rabbit as well as the anterior pituitary of rat, bovine, and hamster, the probe covalently labels a peptide of Mr= 94,000. Specificity of the labeling is typically D2‐‐dopaminergic in character. The covalent labeling is blocked by (+)‐butaclamol but not by the inactive (‐) isomer. Agonists block incorporation with the order of potency: N‐n‐propylnorapomorphine > apomorphine > dopamine. The D2‐‐selective antagonist spiperrone blocks labeling of the Mr= 94,000 peptide whereas the D1‐‐selective antagonist SCH‐23390 is ineffective. Thus, these results indicate that the ligand binding subunit of the D2‐‐dopamine receptor resides on a Mr= 94,000 peptide in these various tissues from several species. Under conditions where proteolysis is not stringently controlled, peptides of lower Mr (32–38,000) are labeled at the expense of the Mr= 94,000 peptide. The most efficient protease inhibitor tested in these systems was EDTA, suggesting that the generation of these lower Mr receptor fragments might be the result of a metal‐dependent proteolysis in the membrane preparations. In the rat neurointermediate lobe, a tissue containing D2‐‐receptors. [125I]N3‐‐NAPS specifically labels a major peptide of Mr± 120,000 in addition to the Mr= 94,000 peptide. This peptide may represent an unprocessed or differently processed form of the receptor or a pharmacologically similar but biochemically distinct form of the receptor in this tissue. Alternatively, the Mr= 94,000 peptide labeled in all the other tissues, even under the conditions used, may already represent a fragment of this high‐Mr peptide.

Solubilization and Characterization of D2-Dopamine Receptors in an Estrone-Induced, Prolactin-Secreting Rat Pituitary Adenoma

Abstract D2‐dopamine (3,4‐dihydroxyphenylethylamine) receptors were successfully solubilized with 3‐[(3‐cholami‐dopropyl)‐dimethylammonio]‐l‐propane sulfonate from an estrone‐induced rat pituitary adenoma. Forty‐five percent of initial protein and 48% of initial [3H]spiroperidol binding sites were solubilized. The high affinity as well as the stereoselectivity of the sites was preserved. The order of potency of dopaminergic agonists was found to be typical of D2 receptors. Target size analysis by radiation inactivation indicated a molecular weight of 143,000 ± 3,000 and of 106,000 ± 4,000 daltons for membrane‐bound and solubilized receptors, respectively. This suggests the loss of a 37,000‐dalton subunit during solubilization without significant modification of binding characteristics. Sodium do‐decyl sulfate‐polyacrylamide gel electrophoresis of receptor protein preparation photolabeled with N‐(p‐azido‐w[125I]‐iodophenethyl)spiroperidol confirmed the existence of a 94,000‐dalton peptide which probably constitutes the li‐gand binding site of the receptor. Thus, our data indicate that chronic estrogen treatment of rats, although inducing a pituitary adenoma, does not modify the pharmacological characteristics of D2 receptors. These data suggest therefore that these adenoma may represent an ideal source of material for further biochemical characterization of D2 receptors.

A novel radioiodinated high affinity ligand for the D2-dopamine receptor: Characterization of its binding in bovine anterior pituitary membranes

A novel high affinity dopaminergic ligand, N‐(p‐aminophenethyl)spiroperidol, has been synthesized and radioiodinated to a specific radioactivity of 2175 . Binding of this ligand to bovine anterior pituitary membranes is: (i) rapid (40–60 min to equilibrium at 25°C) and reversible t = 1 h at 25°C); (ii) saturable and of high affinity (K D ~ 20 pM) and (iii) displays a typical D2‐dopaminergic specificity. The ligand, which identifies the same number of receptor sites as other tritiated antagonist ligands, can be used in different tissues and preparations to delineate the characteristics of the D2 receptor. Thus, this high affinity, high specific radioactivity ligand (N‐(p‐amino‐m‐[125I]iodophenethyl)spiroperidol) represents a tool which until now had not been available for the characterization of the D2‐dopamine receptor.

Biochemical properties of D1 and D2 dopamine receptors.

The physiological action of dopamine are mediated by two distinct subtypes of receptors, D1 and D2 dopamine receptors. D1-receptors are linked to stimulation of adenylate cyclase whereas D2-receptors inhibit the enzyme and may also couple to other signal transduction systems such as ion channels. In order to characterize these receptors at the biochemical level we have developed specific probes for the identification and purification of these proteins. The ligand binding sites of the two receptors have been identified by photoaffinity labeling and reside on distinct polypeptides. In rat striatum, the D1 receptor binding site can be identified as a peptide of Mr = 72,000. In contrast, the D2 receptors appears to reside on an Mr = 94,000 peptide in most tissues. A larger peptide of Mr = 120,000 identified in the intermediate lobe of pituitary may represent the unproteolyzed form of this receptor. An affinity chromatography purification procedure has been developed for the D2 dopamine receptor. This procedure affords a substantial purification (greater than 1000 fold) of the receptor solubilized from bovine anterior pituitary glands with complete retention of its binding properties. These biochemical tools should eventually lead to the complete characterization of these two receptor subtypes.

Regulation of the Adenylate Cyclase Signalling Pathway: Potential Role for the Phosphorylation of the Catalytic Unit by Protein Kinase A and Protein Kinase C

We have investigated the phosphorylation of the pure catalytic unit of adenylate cyclase by cyclic AMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC). The catalytic unit of adenylate cyclase from bovine striatum was purified to apparent homogeneity by sequential affinity chromatography on forskolin-Sepharose and wheat germ aggulutinin-agarose to a specific activity of 1.5 μmol.mg-1.min-1. The enzyme migrates as a single band of M ~160,000 on sodium dodecyl sulfate-polyacrylamide electrophoresis gels and co-elutes with adenylate cyclase activity on steric-exclusion HPLC. The purified catalytic unit can be co-reconstituted with purified β2-adrenergic receptor and stimulatory guanine nucleotide regulatory protein (GS) resulting in their functional coupling. The enzyme can be phosphorylated by both PKA and PKC up to 0.9 mol of phosphate/mol of enzyme. Phosphorylation of the catalytic unit by PKA reduces the Gpp(NH)p-stimulated activity of the enzyme by 30% when co-reconstituted with GS, whereas PKC-phosphorylation of the enzyme enhances this activity by 25%. These results suggest that hormone-sensitive adenylate cyclase systems may be regulated in vivo by PKA- and PKC-dependent phosphorylation of their catalytic units.

D1 and D2 Dopamine Receptors: Identification by Photoaffinity Labeling and Purification by Affinity Chromatography

In an attempt to characterize the receptors which mediate the physiological actions of dopamine, we have developed several probes for the characterization of both the D1 and D2 subtypes of dopamine receptors. To identify the ligand binding site of these receptors we have developed affinity and photoaffinity probes based on the structure of high affinity selective antagonists. [125I]p(azidcphenethyl) spiperone has been used to identify the ligand binding site of the D2 dopamine receptor in several tissues (Amlaiky and Caron, 1985). More recently, our laboratory has also characterized the ligand binding subunit of the D1 dopamine by covalently incorporating a radioiodinated arylamine derivative of the selective D1 antagonist SCH-23390 into rat striatal membranes by photoaffinity crosslinking (Amlaiky et al. 1987). Another derivative of the potent D2 antagonist spiperone (carboxynethyleneoximinospiperone) has been used in the development of an affinity chromatography procedure for the purification of the D2 receptor from bovine anterior pituitary gland (Senogles et al., 1986).