Production of Polyclonal Antibody to the Bovine Adrenal Atrial Natriuretic Factor-R1 Receptor

Abstract

A polyclonal antibody monospecific for an intracellular epitope of the atrial natriuretic factor (ANF)-R₁ receptor was produced. The receptor protein (200 pmoles) was purified to homogeneity from bovine adrenal zona glomerulosa (BAZG), reduced, alkylated and digested with trypsin. The tryptic fragments were purified by reverse-phase h.p.l.c. on a C18 column. Based on the sequence of one of these fragments, a peptide was chemically synthesized, coupled to thyroglobulin and injected into rabbits. The antibody obtained was shown to be specific for the R₁-type as no receptor was detected in bovine red blood cells (RBC) (which are devoid of ANF receptors) and in NIH-3T3 cell membranes (where only the R₂-type is expressed). Several other tissues were screened and comparison of the immunoreactive receptor density estimates with those obtained by ANF binding yielded a correlation coefficient (r²) of 0.965. The minimal detectable dose was typically 3 fmoles/tube and the ED⁵⁰ of the RIA was 30 fmoles/tube. Cyanogen bromide digestion of the receptor was essential for antigenic detection, indicating that the epitope is probably hindered due to the tertiary structure of the native protein. Moreover, location of the epitope in the kinase homology domain of the receptor, combined with partial tryptic digestion, suggests that the proteolysis-sensitive region of the receptor is located between the transmembrane- spanning domain and the amino acid 586. This method of production of antibodies should be useful to precisely map the amino acids involved in various functions of the receptor.