Quantitation of isotypes of D2 receptors using solution hybridization

Solution hybridization was used to quantitate the expression of the long and short splice variants of mRNA coding for the dopamine D2 receptor. Sequences corresponding to 450 bases of the coding region of the long form of the third intracellular loop and the full-length coding sequences of the long and short isoforms of the D2 receptor were amplified using PCR and cloned into a Bluescribe (pBS+) vector. Phage polymerases were used to synthesize riboprobes complementary to the third intracellular loop. Hybridization was carried out using sense strand RNA or total RNA isolated from tissue or cells. After hybridization and digestion with nucleases, the hybridization products were size-fractionated on a urea acrylamide gel. The RNA coding for the D2 receptor appeared as two distinct bands. One band (438 base pairs) represents the long form of the receptor and a second band (285 base pairs) represents the short form of the RNA coding for the receptor. Quantitation of the amount of RNA present suggests that in the striatum the long form of RNA coding for the receptor is expressed at higher levels than is the short form. In a cell line containing dopamine receptors derived from the pituitary (SUPI), the long form predominates to an even greater extent than in the striatum.