cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, smooth muscle myosin light-chain kinase, and phosphorylase kinase were examined with respect to their ability to phosphorylate porcine atrial muscarinic receptors (mAcChRs). Experiments were performed both in detergent solution and in a reconstituted system containing the mAcChR alone or in the presence of the purified porcine atrial inhibitor guanine nucleotide binding protein (Gi). Only cAMP-dependent protein kinase was capable of phosphorylating the receptor under any of the experimental conditions examined. Phosphorylation of the mAcChR in the detergent-solubilized state resulted in a loss of ligand binding sites that was reversible upon treatment with calcineurin in the presence of calcium and calmodulin. Upon reconstitution, the apparent stoichiometry of phosphorylation was increased by about 15-fold. Carbachol-stimulated covalent incorporation of phosphate was found only in the reconstituted system in the presence of Gi, suggesting that the large agonist-stimulated increase in phosphorylation observed in vivo [Kwatra, M. M., & Hosey, M. M. (1986) J. Biol. Chem. 261, 12429-12432] may in part result from a unique receptor conformation that occurs upon association with this protein. Ligand binding studies indicated that phosphorylation of the mAcChR in the detergent-solubilized or reconstituted state did not affect its interaction with carbachol or L-quinuclidinyl benzilate in vitro. Carbachol-induced stimulation of the GTPase activity of Gi in the reconstituted system was also unaffected by phosphorylation.

A monoclonal antibody (mAb L6) to a smallcell lung carcinoma surface antigen recognizes a common epitope of vasopressin-neurophysin and oxytocin-neurophysin in hypothalamic nuclei. We now report on the identification of a neurophysin-like precursor in human lung carcinoma (LX-1) cell membrane. mAb L6 immunoaffmity chromatography of solubilized membranes resulted in a single band of =45 kDa.Western blot analysis demonstrated immunoreactivity of this band with mAb L6, anti-vasopressin, and an antibody to the vasopressin precursor, pro-pressophysin. N-terminal sequencing of this band demonstrated a 21-amino acid homology with the N terminus of human pro-pressophysin, and substitution of a Cys33 residue in the tumor antigen with Arg33. Absence of immunoreactivity with the antibodies described above in cytosolic extracts and culture medium suggests nonsecretion of processed or intact pro-pressophysin-like peptide. Northern analysis of LX-1 mRNA with a 30-mer to the C terminus of rat pro-pressophysin resulted in a band of =4000 base pairs, 250 base pairs larger than hypothalamic message. In situ hybridization of LX-1 tumor-bearing nude rat brain with the same probe demonstrated specific hybridization in rat hypothalamus and xenografted tumor. These findings suggest expression of a pro-pressophysin-like protein in this tumor cell line that is preferentially targeted to the cell membrane.Lung carcinomas are well known for their association with paraneoplastic syndromes, often through production and secretion of peptide hormones. Hormones such as vasopressin (VP) (1), neurophysin (NP) (2), and bombesin (3) can stimulate growth of tumor cells by functioning as autocrine growth factors. Elevated plasma levels of tumor-secreted hormones have been documented in patients with small-cell lung carcinomas and thus may allow early detection of tumor and monitor efficacy of treatment (4).We have been characterizing a monoclonal antibody generated against a human lung adenocarcinoma (mAb L6) that binds to a surface epitope of human lung, colon, breast, and ovarian carcinomas (5). Our studies demonstrated that mAb L6 specifically recognizes a common domain within vasopressin-neurophysin (VP-NP) and oxytocin-neurophysin (OT-NP) (6). Since mAb L6 is immunoreactive with a membrane-bound protein, it was of interest to determine whether the mAb L6-reactive surface antigen was NP or NP related.We now report on the identification of a NP-like precursor isolated from the human lung cancer cell line, LX-1 (7). This tumor cell line does not appear to process or secrete this precursor, being targeted instead to the cell membrane. To our knowledge no one else has reported a neuroendocrine hormone precursor being preferentially expressed in cell membranes. Antibodies. mAb L6 is an IgG2. mAb that recognizes a cell-surface antigen in human lung, breast, colon, and ovarian carcinomas (5) as well as specifically recognizing a common domain of both VP-NP and OT-NP (6). P1.17, an IgG2a mAb, was used as a control in the blotting experiments. Y...

Mouse monoclonal antibody (mAb) L6 identifies an antigen expressed on the cell surface of many different human carcinomas. While studying the binding activity of mAb L6 to intracerebral tumor xenografts of human lung carcinoma LX-1 cells in nude rats using immunohistological techniques, we observed that L6 can also bind to a cytoplasmic antigen expressed in the magnocellular component of the hypothalamo-neurohypophysial system. Double-labeling experiments with antisera to vasopressin and oxytocin confirmed the localization of L6 immunoreactivity within both peptide-containing cell groups. L6 immunoreactivity in Brattleboro rats (with genetic deletion in the vasopressin gene) was exclusively localized within oxytocin neurons. Oxytocin and vasopressin failed to block L6 staining which suggested that its target epitope resides within the neurophysin sequence, and this explanation was supported by the finding that adsorption of L6 with porcine neurophysin completely eliminated hypothalamic immunoreactivity. Western blot analysis of bovine neurophysin and human pituitary extracts identified L6-immunoreactive bands which corresponded to the position of neurophysin and pro-pressophysin, confirming that L6 immunoreactivity in hypothalamus is related to neurophysin. Thus, monoclonal antibody L6, which is highly reactive with a membrane antigen of human lung cancer cell line LX-1 recognizes a cytoplasmic epitope in hypothalamic neurons identified as neurophysin by immunohistochemistry and Western analysis.

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A patient with a primary undifferentiated carcinoma of the nasopharynx manifested the clinical syndrome of inappropriate antidiuretic hormone secretion (SIADH). Immunohistochemical techniques demonstrated the presence of vasopressin, neurophysin, and their precursor (propressophysin) in the cancer cells. In situ hybridization additionally confirmed the expression of propressophysin messenger RNA in these cells. To the knowledge of the authors, this represents not only the first case of SIADH caused by carcinoma of the nasopharynx, but also the first report of pathologic confirmation of the syndrome with the use of both molecular and immunologic probes. Cancer 1992; 69:1315‐1319.