Development of a Site-Directed Polyclonal Antibody against the Pituitary Growth Hormone-Releasing Hormone Receptor and Its Use to Estimate GHRH Receptor Concentration in Normal and Hypothyroid Rats

Abstract

A site-directed polyclonal antipeptide antibody was generated in rabbits against segment 392–404 of the rat pituitary growth hormone-releasing hormone receptor (GHRH-R), using a multiple antigenic peptide system strategy of immunization. This C-terminal intracellular region of the rat GHRH-R exhibits 85% sequence identity with the human GHRH-R. The purified anti-GHRH-R(392–404) IgGs were characterized in cell lines expressing the human GHRH-R and in rat and human anterior pituitary, using immunoblotting. The polyclonal antibody recognized a 45-kD protein in human GHRH-R-transfected BHK 570 cell membrane preparations but not in wild-type cells. A 45-kD N^α-tagged human GHRH-R was immunodetected with both antitag and anti-GHRH-R antibodies in human GHRH-R-transfected HEK 293 cells. Cross-linking of [¹²⁵I-Tyr¹⁰]hGHRH(1–44)NH₂ to GHRH-R-transfected BHK cells led to the detection of a major and specific 45-kD radioactive complex. Its probing with the anti-GHRH-R(392–404) IgGs led also to the detection of a 45-kD entity. In rat anterior pituitary homogenates or membrane preparations, immunoblotting led to the detection of 44-, 47- and 65-kD proteins. In human anterior pituitary membrane preparations, immunoblotting led to the detection of 52- and 55-kD proteins. No immunoreactive signal was observed in the rat liver. Cross-linking of [¹²⁵I-Tyr¹⁰]hGHRH(1–44)NH₂ to rat anterior pituitary homogenates revealed the presence of specific 28-, 47- and 65-kD radioactive complexes. Probing of these radioactive complexes with the anti-GHRH-R(392–404) IgGs resulted in the visualization of 28-, 47- and 65-kD entities and of an additional immunoreactive 44-kD protein. To assess the usefulness of this GHRH-R antibody, estimation of changes in the concentration of rat anterior pituitary GHRH-R was performed by immunoblotting and compared to binding data after a 3-week antithyroid treatment. The treatment known to depress the 2.5- and 4-kb GHRH-R mRNA transcripts by at least 1.7-fold decreased the apparent maximal concentration of high (B_max1) and low (B_max2) affinity binding sites by 4.6- and 15.2-fold, respectively, and the 47- and 65-kD GHRH-R proteins by 3.5- and 1.25-fold, respectively. Altogether, the characteristics of the anti-GHRH-R(392–404) polyclonal antibody indicate that it specifically recognizes the human and rat GHRH-R. It also represents an additional valuable tool to estimate variations of GHRH-Rs in physiopathological conditions known to affect GHRH-R mRNA and/or GHRH binding site concentrations.