Replacement of Serine363 and Serine375 Codons by Alanine in Rat μ-Opioid Receptor cDNA

Abstract

The aim of this study was to use site directed mutagenesis technique to construct a vector in which serine³⁶³ and serine³⁷⁵ residues of the COOH-terminal portion of the μ-opioid receptor (MOR) were substituted by alanine. These constructs are essential in studying G-protein coupled receptor kinase-mediated MOR desensiti-zation. The nested PCR carried out for conversion of serine³⁶³ and serine³⁷⁵ to alanine resulted in the production of a band comparable to the expected size of 1400 bp. Restriction analysis of these bands confirmed the integrity of the PCR products. Ligation of the mutated PCR product into pcDNA3 and its digestion with appropriate restriction enzymes further confirmed the integrity of the PCR product and its orientation into the vector.