Complementary Peptides as Probes to Explore Neuropeptide Receptors on Lymphocytes

Abstract

Studies on neuroendocrine hormone receptor have been hampered by low numbers and concentrations of receptors found within and outside the neuroendocrine system. The complementary peptide approach is particularly useful for dealing with this problem and has been used to characterize lymphoid receptors for arginine vasopressin (AVP), corticotropin (ACTH), substance P, and opioid peptides. A nonapeptide derived by reading of the complementary DNA strand of the bovine AVP gene in the 3′ to 5′ direction specifically blocks the AVP helper signal for interferon-γ production by mouse T lymphocytes. Antibodies to 3′-5′ AVP-binding peptide bound to cells, and the binding was inhibited by excess AVP. Thus, binding of anti-3′-5′ AVP-binding peptide antibodies to the AVP receptor was specific. The complementary peptide approach has also been used to produce antibodies specific for the ACTH receptor complex. Complementary peptides to ACTH derived by reading in either the 5′ to 3′ or 3′ to 5′ direction were able to bind to ACTH. Mono-specific antibodies to the ACTH(1-24) complementary peptide caused an ACTH-like steroidogenic response of cultured mouse adrenal cells, presumably by binding to the ACTH receptor, and binding was specifically inhibited by ACTH. The ACTH receptor complex from solubilized adrenal cells was shown to consist of four subunits with M_r 83,000, 64,000, 52,000, and 22,000. The 83,000 and 52,000 M_r subunits are disulfide linked and noncovalently associated with the other subunits, with binding of labeled ACTH localized to the 83,000 M_r subunit. Similarly, a complementary peptide was shown to bind directly to substance P in a saturable and dose-dependent manner. Affinity-purified antibodies to the substance P complementary peptide directly bound to IM-9 cells and blocked the binding of substance P to its IM-9 receptor. Further, these antibodies recognized a single protein of 58 kDa from solubilized IM-9 cells. Complementary peptides have been used to characterize opioid receptors, and studies show that antigenic and functional similarity exists between opioid receptors from brain and lymphocytes. Thus, the complementary peptide approach can be used to produce antibodies specific for neuroendocrine receptors, which are useful tools for the isolation and characterization of these receptors.