Search

Thrombopoietin (TPO) is a hematopoietin important for megakaryocyte proliferation and production of blood platelets. We sought to characterize how TPO binds and activates its receptor, myeloproliferative leukemia virus receptor. The erythropoietin-like domain of TPO (TPO1-153) has been fused to the gIII coat protein of M13 bacteriophage. Forty residues were chosen for mutation to alanine using the criteria that they were charged residues or predicted to be solvent-exposed, based on a homology model. Phage enzyme-linked immunosorbent assay was used to determine affinities for binding to both the TPO receptor and five anti-TPO1-153 monoclonal antibodies. Mutations at mostly positively charged residues (Asp8, Lys14, Lys52, Lys59, Lys136, Lys138, Arg140) caused the greatest reduction in receptor-binding affinity. Most of these residues mapped to helices-1 and -4 and a loop region between helix-1 and helix-2. Two of the monoclonal antibodies that blocked TPO binding and bioactivity had determinants in helix-4. In contrast, the other three monoclonal antibodies, which were effective at blocking TPO activity but did not block initial binding of TPO to its receptor, had epitopes predominantly on helix or 3. These results suggest that TPO has two distinct receptor-binding sites that function to dimerize TPO receptors in a sequential fashion.

The most potent known agonist for the natriuretic peptide receptor-B (NPR-B)/guanylyl cyclase-B is C-type natriuretic peptide (CNP). A homologous ligand-receptor system consists of atrial natriuretic peptide (ANP) and NPR-A/guanylyl cyclase-A. A third member of this family is NPR-C, a non-guanylyl cyclase receptor. Monoclonal antibodies were raised against NPR-B by immunizing mice with a purified receptor-IgG fusion protein consisting of the extracellular domain of NPR-B and the Fc portion of human IgG-gamma 1. One monoclonal antibody, 3G12, did not recognize NPR-A or NPR-C and bound to human and rat NPR-B. CNP binding to NPR-B and stimulation of cGMP synthesis were inhibited by 3G12. With cells isolated from either the media or adventitia layers of rat thoracic aorta, 3G12 did not interfere with ANP-stimulated cGMP synthesis, but it inhibited CNP-stimulated cGMP levels in cells from both layers. CNP (IC50 = 10 nM) and ANP (IC50 = 1 nM) caused relaxation of phenylephrine-contracted rat aortic rings. 3G12 caused a marked increase in the IC50 for CNP, from 10 nM to 140 nM, but failed to affect ANP-mediated relaxation. Therefore, our results for the first time demonstrate that CNP relaxes vascular smooth muscle by virtue of its binding to NPR-B.

The heregulin/neu differentiation factor gene products were purified and cloned based on their ability to stimulate the phosphorylation of a 185-kDa protein in human breast carcinoma cell lines known to express erbB2. However, not all cells that express erbB2 respond to heregulin, indicating that other components besides erbB2 may be required for heregulin binding. Cells that are transfected with the closely related receptor, erbB3, display a single class of lower affinity heregulin binding sites than has been previously observed on breast carcinoma cell lines. Little or no stimulation of tyrosine phosphorylation in response to heregulin occurs in cells that are transfected with erbB3 alone. Transfection of cells with erbB3 and erbB2 reconstitutes a higher affinity binding receptor, which is also capable of generating a tyrosine phosphorylation signal in response to heregulin. A monoclonal antibody to erbB2 will inhibit heregulin activation of tyrosine phosphorylation and binding in cells transfected with both receptors but not with erbB3 alone. In cells expressing erbB2 and erbB3, both proteins become tyrosine-phosphorylated upon interaction with heregulin. Direct interaction between heregulin and the two proteins was demonstrated by chemical cross-linking experiments using 125I-heregulin followed by immunoprecipitation with antibodies specific for erbB2 or erbB3.

Most of the physiological actions of atrial natriuretic peptide (ANP) may be attributed to activation of the natriuretic peptide receptor-A (NPR-A) guanylyl cyclase. We report here that truncation of the NPR-A cytoplasmic domain results in increased expression of cell surface ANP binding sites. The truncated receptor exhibited a hyperbolic time course for ANP binding and had a high affinity for [125I]hANP, Kd = 8 pM. Cells expressing truncated NPR-A were used as an immunogen to obtain monoclonal antibodies against the native conformation of the extracellular domain. These antibodies were used to select for high levels of stable NPR-A expression in 293 cells, by fluorescence-activated cell sorting. Disuccinimidyl suberate cross-linked [125I]ANP to 135-kDa NPR-A on intact cells. Monoclonal antibody immunoprecipitation of 35S-labeled proteins revealed NPR-A size heterogeneity, with 135- and 125-kDa species. A synthetic peptide antibody directed against the extracellular domain immunoprecipitated 125-kDa NPR-A, but recognized both sizes of receptor by Western blotting. The 125-kDa NPR-A did not bind to or cross-link ANP. NPR-A size variants were expressed on the cell surface, and heterogeneity was removed by deglycosylation with protein:N-glycosidase F. Our results suggest that the degree of N-linked glycosylation of the NPR-A extracellular domain influences the ability to bind ANP.

An expression plasmid encoding the human 75-kDa tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable human cell line (293/TNF-R2) overexpressing TNF-R2. Ligand binding analysis revealed high affinity binding (Kd = 0.2 nM) with approximately 94,000 +/- 7,500 sites/cell for 125I-TNF-alpha and approximately 5-fold lower affinity for TNF-beta (Kd = 1.1 nM) with 264,000 +/- 2,000 sites/cell. Cross-linking of 125I-TNF-alpha and 125I-TNF-beta to 293/TNF-R2 cells yielded predominant complexes with apparent molecular weights of 211,000 for TNF-alpha and 205,000 and 244,000 for TNF-beta, suggesting these complexes contain two or three TNF-R2 molecules. Immunoprecipitation of TNF-R2 from 32P-labeled 293/TNF-R2 cells demonstrated that the receptor is phosphorylated. The majority (97%) of 32Pi incorporation was found in serine residues with a very low level of incorporation (3%) in threonine residues. TNF-alpha treatment of 293/TNF-R2 cells did not significantly affect the degree or pattern of phosphorylation. Cell surface-bound 125I-TNF-alpha was slowly internalized by the 293/TNF-R2 cell line with a t1/2 = 25 min. Shedding of the extracellular domain of TNF-R2 was induced by 4 beta-phorbol 12-myristate 13-acetate but not by TNF-alpha or TNF-beta.

The extracellular portion of the type 1 (p55) and type 2 (p75) tumor necrosis factor (TNF) receptors contains a repetitive amino acid sequence pattern of four cysteine-rich domains (CRDs). This pattern is found also in several other cell surface proteins, including the p75 nerve growth factor receptor and the CD40, 4-1BB, OX40, Fas, and CD27 antigens. To investigate whether CRDs play a role in TNF binding, we have constructed soluble variants of the extracellular portion of human type 1 TNF receptor (sTNFR1), in which CRD1 (N-terminal) or CRD4 (C-terminal) was deleted by mutagenesis. These variants or a wild type sTNFR1 were linked in their C terminus to the hinge and Fc portion of IgG1 heavy chain to create sTNFR1-IgG chimeras (immunoadhesins). Deletion of either CRD1 or -4 did not cause any major perturbations in the structure of the sTNFR1 variants, as evidenced by their efficient expression and secretion from transfected cells, and by their binding to conformation-dependent monoclonal antibodies that recognize diverse epitopes on sTNFR1. The wild type sTNFR1 immunoadhesin exhibited high affinity binding to TNF alpha (Kd = 65 pM) and TNF beta (Kd = 640 pM). Deletion of CRD4 resulted in about a 10-fold reduction in affinity for TNF alpha (Kd = 660 pM) and for TNF beta (Kd = 5.7 nM). In contrast, deletion of CRD1 resulted in a complete loss of binding to TNF alpha and to TNF beta. These results indicate that CRD4 is important but not necessary for TNF binding, while CRD1 is required. In addition, the results suggest some overlap between the TNFR1 binding sites for TNF alpha and TNF beta, despite low amino acid sequence homology between these cytokines.