RESUMEN
The LH/hCG receptor is a G protein-coupled receptor with an N-terminal extracellular domain involved in hormone-receptor interaction. The recombinant porcine receptor, stably expressed in Chinese hamster ovary (CHO) cells, has the same characteristics (Kd and cAMP production) as in Leydig cells. Six synthetic peptides derived from the receptor ectodomain and two polyclonal anti-peptide sera were tested in the homologous system porcine LH and porcine LH receptor. Their ability to inhibit hormone binding and signal transduction on CHO cells expressing the recombinant receptor was evaluated. Peptides 25-40 and 107-121 exhibited a high transduction inhibition as compared with hormone binding, peptides 21-36, 102-111, and 102-121 inhibited hormone binding more efficiently than signal transduction, and peptide 7-24 exhibited inhibition of both hormone binding and hormone-induced cAMP production. Immunoglobulins against peptides 21-36 and 102-111 inhibited both hormone binding and receptor activation suggesting that these sequences are located on the receptor surface. The data suggest that multiple, discontinuous regions of the extracellular domain of porcine LH receptor are involved in hormone binding and signal transduction. Two minimum critical sequences, 21-24 and 102-107, are involved in hormone binding and vicinal segments may be implicated in signal transduction.
Asunto(s)
Hormona Luteinizante/metabolismo , Estructura Secundaria de Proteína , Receptores de HL/química , Receptores de HL/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/farmacología , Células CHO , Secuencia de Consenso , Cricetinae , AMP Cíclico/metabolismo , Ensayo de Inmunoadsorción Enzimática , Exones , Cinética , Hormona Luteinizante/farmacología , Modelos Estructurales , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Ratas , Receptores de HL/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Porcinos , TransfecciónRESUMEN
Polyacrylamide-based solid-phase supports were used in the production of a synthetic peptide analogous to amino acid sequence 1-9 from bovine aortic actin. Different peptidyl-resins were prepared and directly used to induce an antibody response in rabbits. Antisera that recognized the resin-bound peptide were compared. Some of them reacted with native aortic actin; this response was highly specific for the smooth muscle alpha actin isotype. We also immunized rabbits with the 1-9 peptide linked to bovine serum albumin. In this case, antisera were less reactive with native aortic actin, and cross-reacted with the two closely related smooth muscle alpha and macrophage actin isotypes. This method provides a rapid and simple procedure for generating peptide immunogens and antibodies to them, by the combination of peptidyl-resin immunizations and resin-bound peptide immunoassays.