Immunocytochemical localisation of plasma membrane GHRH receptors in human tumours using a novel anti-peptide antibody.
Antagonists of growth hormone releasing hormone (GHRH) directly inhibit the growth of a variety of human neoplasms. However, the plasma membrane receptor mediating these effects has not been immunocytochemically visualised in primary tumour cells. Given that previous attempts using an antibody to the amino-terminal region did not result in the visualisation of plasma membrane receptors, we have developed and characterised an anti-peptide antibody to the carboxy-terminal region 403-422 of the human pituitary GHRH receptor. This sequence is identical to residues 339-358 of splice variant 1 (SV1) of tumoural GHRH receptors. Specificity of the antibody was demonstrated by (1) immunocytochemical staining of GHRH receptor-transfected cells, (2) detection of a broad glycosylated protein band migrating at Mr 50,000-60,000 in Western blots of membranes from human pituitary, and (3) abolition of tissue immunostaining by preadsorbtion of the antibody with its immunising peptide. The distribution of GHRH receptors was investigated in 69 formalin-fixed, paraffin-embedded human tumours showing that GHRH receptors were frequently expressed in breast, ovarian and prostate carcinomas. Immunoreactive GHRH receptors were clearly confined to the plasma membrane and uniformly present on nearly all tumour cells. In Western blots of membranes prepared from human tumours, the anti-GHRH receptor antibody detected a non-glycosylated protein band migrating at Mr 40,000, which corresponds to the expected molecular weight of splice variant 1 of tumoural GHRH receptors. Together, our findings provide direct evidence for the presence of GHRH receptor protein on the plasma membrane of primary human tumour cells. The GHRH receptor visualisation could be of value for a rapid immunohistochemical identification of those tumours which could be a target for diagnostic or therapeutic intervention using GHRH analogues.
Schulz
Solveig
S
Department of Obstetrics and Gynecology, Otto-von-Guericke-University, 39120 Magdeburg, Germany.
Röcken
Christoph
C
Schulz
Stefan
S
D000906:Antibodies / Q000032:analysis*; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D009363:Neoplasm Proteins / Q000276:immunology / Q000378:metabolism*; D009369:Neoplasms / Q000175:diagnosis*; D010455:Peptides / Q000276:immunology*; D018013:Receptors, Neuropeptide / Q000276:immunology / Q000378:metabolism*; D018017:Receptors, Pituitary Hormone-Regulating Hormone / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D009363:Neoplasm Proteins; D010455:Peptides; D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; C048250:somatotropin releasing hormone receptor