Immunocytochemical localization of prohormone convertases PC1 and PC2 in the mouse thyroid gland and respiratory tract.

We examined immunocytochemical localization of the prohormone convertases, PC1 and PC2, in the thyroid gland and respiratory tract of the adult mouse using the indirect enzyme- and immunogold-labeled antibody methods for light and electron microscopy, respectively. In the thyroid gland, PC1- and/or PC2-immunoreactive cells were cuboidal, scattered in the follicular epithelium and in the interfollicular spaces. When serial sections were immunostained with anti-calcitonin, anti-PC1, anti-calcitonin-gene-related-peptide (CGRP), and anti-PC2 sera, respectively, localization of both PC1 and PC2 was restricted to the calcitonin/CGRP-producing parafollicular cells. In the respiratory tract, only PC1 immunoreactivity was observed in the basal granulated neuroendocrine cells, which were scattered in the tracheal epithelium. Consecutive sections immunostained with anti-PC1 and anti-CGRP sera showed that a subpopulation of these PC1-immunoreactive cells contained CGRP. Double immunogold electron microscopy of the thyroid parafollicular cells revealed that calcitonin- and/or CGRP-immunopositive secretory granules were also labeled with both PC1 and PC2. These findings suggest that procalcitonin is proteolytically cleaved by PC2 alone or by PC2 together with PC1, and that the proCGRP is cleaved by PC1.     

Developmental expression of otoconin-22 in the bullfrog endolymphatic sac and inner ear.

In amphibians, calcium carbonate crystals are present in the endolymphatic sac and the inner ear. The formation of these crystals is considered to be facilitated by a protein called otoconin-22. We examined the spatial and temporal expression of otoconin-22 during the development of the bullfrog (Rana catesbeiana) using RT-PCR, in situ hybridization (ISH), and immunofluorescence techniques. By RT-PCR, otoconin-22 mRNA was first detected in embryos at Shumway stage 20, and this expression pattern continues in late stages. The first otoconin-22 mRNA-positive reaction was detected in stage 22 embryos in the placode of the endolymphatic sac. Otoconin-22 protein was observed in the epithelial cells of the endolymphatic sac at stage 24. On the other hand, a whole-mount ISH technique showed the first expression of otoconin-22 mRNA in the inner ear, in addition to the endolymphatic sac, at the mid-phase of Shumway stage 25. We discuss the role of otoconin-22 in the formation of calcium carbonate crystals in the endolymphatic sac and inner ear.     

CCK processing by pituitary GH3 cells, human teratocarcinoma cells NT2 and hNT differentiated human neuronal cells evidence for a differentiation-induced change in enzyme expression and pro CCK processing

Human teratocarcinoma Ntera2/c 1.D1 (NT2) cells express very low levels of the prohormone convertase enzyme PC1, moderate levels of PC2 and significant levels of PC5. When infected with an adenovirus which expresses rat CCK mRNA, several glycine-extended forms were secreted that co-eluted with CCK 33, 22 and 12. Amidated CCK is not produced because these cells appear to lack the amidating enzyme. Pituitary GH3 cells express high levels of PC2 and PC5. CCK adenovirus-infected GH3 cells secrete amidated versions of the same peptides as NT2 cells. Differentiation of NT2 cells into hNT cells with retinoic acid and mitotic inhibitors increased expression of PC5 and decreased expression of PCI and PC2. CCK adenovirus-infected differentiated hNT cells also secrete glycine extended CCK products and the major molecular form produced co-eluted with CCK 8 Gly. These experiments demonstrate that the state of differentiation of this neuronal cell line influences its expression of PC 1,2, and 5 and its cleavage of pro CCK and suggests that these cells may make an interesting model to study how differentiation alters prohormone processing. These results also support the hypothesis that PC5 in differentiated neuronal cells is capable of processing pro CCK to glycine-extended CCK 8.     

Molecular cloning and expression of prohormone convertases PC1 and PC2 in the pituitary gland of the bullfrog, Rana catesbeiana

We cloned cDNAs encoding PC1 and PC2 from a cDNA library constructed for the anterior pituitary gland of the bullfrog (Rana catesbeiana) and sequenced them. The bullfrog PC1 cDNA consisted of 2972 base pairs (bp) with an open reading frame of 2208 bp and encoded a protein of 736 amino acids, including a putative signal peptide of 26 amino acids. The protein showed a high homology to R. ridibunda PC1 (95.1%) and mammalian PC1 (72.6%). The bullfrog PC2 cDNA consisted of 2242 bp with an open reading frame of 1914 bp and encoded a protein of 638 amino acids, including a putative signal peptide of 23 amino acids. This protein showed a high homology to R. ridibunda PC2 (95.5%) and mammalian PC2 (84.8%). The catalytic triad of serine proteinases of the subtilisin family was found at Asp-168, His-209, and Ser-383 in the PC1 protein and at Asp-167, His-208, and Ser-384 in the PC2 protein. In situ hybridization staining revealed that PC2 mRNA was detected in corticotrope cells of the tadpoles, but not in those of the adults. In the adult, only PC1 mRNA was detected in the pars distalis but both PC1 and PC2 mRNAs were detected in the pars intermedia. The data also showed that PC1 mRNA was expressed in gonadotrope cells.     

Gene expression patterns of pro-opiomelanocortin-processing enzymes PC1 and PC2 during postnatal development of rat corticotrophs.

We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the anterior pituitary cells of developing rats by a double staining procedure using in situ RT-PCR and an immunofluorescence technique. In the adult, both PC1 mRNA and PC2 mRNA were expressed in corticotrophs, gonadotrophs, thyrotrophs, and mammotrophs. These cells, except for corticotrophs, had previously been considered to be ones in which proprotein processing does not take place, but both PC1 and PC2 may be necessary to process other proteins, such as granin family proteins, having proteolytic cleavage sites and located in secretory granules of the above trophs. In addition, no PC1 or PC2 mRNA was expressed in somatotrophs, which is consistent with the fact that somatotrophs do not contain these granins. In addition, 7B2 mRNA was expressed in these PC2-positive trophs, suggesting that there is a functional relationship between PC2 and 7B2 proteins. We found that alpha-MSH was expressed in the corticotrophs of the postnatal rat and that the number of alpha-MSH-immunopositive corticotrophs decreased as development proceeded. Because the changes in the pattern of POMC processing are considered to depend on the relative expression levels of PC1 and PC2, PC1 and PC2 mRNAs were examined in corticotrophs during postnatal development. We found a decrease in the number of PC2 mRNA-positive cells, which coincided with one in the number of alpha-MSH-immunopositive corticotrophs, as postnatal development proceeded. Our present data demonstrate that the alpha-MSH production varies directly in accordance with the expression of PC2. We also discuss the possible significance of alpha-MSH production during the postnatal period.     

Molecular Characterization and Differential Gene Induction of the Neuroendocrine-Specific Genes Neurotensin, Neurotensin Receptor, PC1, PC2, and 7B2 in the Human Ocular Ciliary Epithelium

Abstract: The ocular ciliary epithelium is a bilayer of neuroepithelial cells specialized in the secretion of aqueous humor fluid and the regulation of intraocular pressure. In this study, we report on the expression of the regulatory peptide neurotensin (NT) and a set of differentiated neuroendocrine markers including neurotensin receptors (NTrs), the prohormone convertases furin, PC1, and PC2, and the neuroendocrine polypeptide 7B2 in the ciliary epithelium. Using a human cell line, ODM-2, derived from the nonpigmented ciliary epithelium, we demonstrate that (1) NT expression is highly activated by nerve growth factor, glucocorticoid, and activators of adenylate cyclase; (2) NTr expression is up-regulated by selective ligand-activated β₂-adrenergic receptor; and (3) PC1 and PC2 expression are up-regulated via distinct signaling transduction pathways. PC1 gene expression is activated by phorbol ester, and PC2 by the same inducers as those of NT expression. A radioimmunoassay for NT detected an NT-like immunoreactivity in human ciliary epithelium and ODM-2 cell extracts, in aqueous humor, and in conditioned culture medium. The results support the view that the entire ciliary epithelium functions as a neuroendocrine tissue, synthesizing, processing, and releasing NT into the aqueous humor where it may exert important physiological functions through autocrine and/or paracrine mechanisms.     

Proteolytic Processing of Chromogranin B and Secretogranin II by Prohormone Convertases

Abstract: Two experimental approaches were used to study the processing of chromogranin B and secretogranin II by prohormone convertases. In GH₃ cells various prohormone convertases were overexpressed together with the substrate chromogranin B by use of a vaccinia virus infection system. PC1 appeared to be by far the most active enzyme and converted chromogranin B to several smaller molecules, including the peptide PE-11. In brain this peptide is cleaved physiologically from chromogranin B. Some processing of chromogranin B and formation of free PE-11 were also observed with PC2 and PACE4. Furin produced larger fragments, whereas PC5-A and PC5-B had negligible effects. As a second model, PC12 cells were stably transfected with PC1 or PC2 to investigate the processing of endogenous chromogranins. Both enzymes effectively cleaved chromogranin B and secretogranin II, liberating the peptides PE-11 and secretoneurin, respectively. However, in transfection experiments the ability to generate the free peptides was more pronounced with PC2 than with PC1. The extent of proprotein processing achieved by prohormone convertases apparently differed depending on the experimental system applied. This suggests that in vivo mechanisms to support and fine-tune the activity of the processing enzymes exist, which might be overlooked by using only one methodological approach.     

Recombinant prohormone convertase 1 and 2 cleave purified pro cholecystokinin (CCK) and a synthetic peptide containing CCK 8 Gly Arg Arg and the carboxyl-terminal flanking peptide

Purified recombinant prohormone convertase 1 and 2 (PC1 and PC2) cleave a peptide containing cholecystokinin (CCK) 8 Gly Arg Arg and the carboxyl-terminal peptide liberating CCK 8 Gly Arg Arg. PC1 and PC2 also cleave purified pro CCK, liberating the amino terminal pro-peptide while no carboxyl-terminal cleavage was detected. Under the conditions of the in vitro cleavage assay, it appears that the carboxyl-terminal cleavage site of pro CCK is not accessible to the enzymes while this site is readily cleaved in a synthetic peptide. Additional cellular proteins that unfold the prohormone may be required to expose the carboxyl-terminal site for cleavage.     

Differential localization of prohormone convertases PC1 and PC2 in two distinct types of secretory granules in rat pituitary gonadotrophs

Prohormone convertases PC1 and PC2 are endoproteases involved in prohormone cleavage at pairs of basic amino acids. There is a report that prohormone convertase exists in the rat anterior pituitary gonadotrophs, where it had previously been considered that proprotein processing does not take place. In addition to luteinizing hormone and follicle-stimulating hormone, rat pituitary gonadotrophs contain chromogranin A (CgA) and secretogranin II (SgII), two members of the family of granin proteins, which have proteolytic sites in their molecules. In the present study we examined whether there is a close correlation between subcellular localization of prohormone convertases and granin proteins. Ultrathin sections of rat anterior pituitary were immunolabeled with anti-PC1 or -PC2 antisera and then stained with immunogold. Immunogold particles for PC1 were exclusively found in large, lucent secretory granules, whereas those for PC2 were seen in both large, lucent and small, dense granules. The double-immunolabeling also demonstrated colocalization of PC2 and SgII in small, dense granules and of PC1, PC2, and CgA in large, lucent granules. These immunocytochemical results suggest that PC2 may be involved in the proteolytic processing of SgII and that both PC1 and PC2 may be necessary to process CgA.     

Human antibodies to phosphocholine. IgG anti-PC antibodies express restricted numbers of V and C regions

We examined the IgG subclass composition and isoelectric focusing (IEF) spectrotype pattern of naturally occurring human IgG antibodies that bind phosphocholine (PC) and found direct evidence for restricted expression of both V and C regions among these antibodies. In most individuals, the isotype of these IgG anti-PC antibodies was primarily IgG2. However, serum from some individuals contained significant amounts of IgG1 and IgG3 anti-PC antibodies. We also found that in individual sera, anti-PC antibodies are pauciclonal, as demonstrated by restricted spectrotypic patterns of the anti-PC antibodies. The IEF pattern of these antibodies were for the most part unique for each individual. In some sera, certain anti-PC antibodies with isoelectric points of basic pH bound PC conjugated to bovine serum albumin (PC-BSA) but did not bind pneumococcal C-carbohydrate bearing PC determinants. In two individuals, we found that the spectrotypes that bound only PC-BSA were of the IgG1 subclass. Taken together, these findings demonstrate that within individual sera, human antibodies to PC are quite restricted in both V and C region expression, and furthermore, these V and C regions of human Ig may not randomly associate.     

Analysis of salmon calcitonin I in the ultimobranchial gland and gill filaments during development of rainbow trout, Oncorhynchus mykiss, by in situ hybridization and immunohistochemical staining

We have cloned two distinct cDNAs encoding salmon-type calcitonin (sCT)-I cDNAs from the ultimobranchial gland of rainbow trout, Oncorhynchus mykiss. Both cDNAs were predicted to encode nearly identical sCT-I precursors which consisted of an N-terminal peptide of 80 amino acid residues, a putative cleavage site Lys-Arg, sCT-I, a cleavage and amidation sequence Gly-Lys-Lys-Arg, and a C-terminal peptide of 18 amino acids. Development of sCT-I-expressing cells was then examined by employing conventional histochemical staining, in situ hybridization with a specific cRNA probe, and further immunohistochemistry. The primordium of the ultimobranchial gland was first identified, as two cell masses, in the region between the alimentary canal and sinus venosus behind the heart 17 days postfertilization (dpf; 14 degrees C). However, expression of sCT-I mRNA could not be detected in this gland until one day later, and appeared at 18 dpf. sCT-I immunoreactivity was first observed at 19 dpf (two days before hatching), and the ultimobranchial gland began to assume a follicular structure at 20 dpf (one days before hatching). As ontogeny proceeded, the sCT-I-immunoreactive cells increased in both number and stainability. The sCT-I mRNA was also expressed on the developing gill filaments, but immunoreactive sCT-I was not detected in these sites. These results provide basic data for further research on the organogenesis of the trout ultimobranchial gland.     

Quantitative comparison of cytokine mRNA and inflammatory responses in cutaneous late phase allergic reactions

The aim of this study was to quantitatively compare expression of mRNA for IL-5 and IFN-gamma with the frequency of mRNA-positive cells, total and activated eosinophils, neutrophils, lymphocytes, and vessels expressing adhesion molecules. Replicate biopsies of skin LPR to pollen antigens (Ag) and control injection sites (B) at 6 and 24 h were assessed for: (1) mRNA for IL-5 and IFN-gamma by quantitative RT-PCR (QC-RT/PCR); (2) frequency of cells expressing mRNA for IL-5 and IFN-gamma by in situ hybridization (ISH); (3) inflammatory cells and adhesion molecule expression. More mRNA for IL-5 was found in Ag- than in B-injected sites at 6 and 24 h by both QC-RT/PCR and ISH. Small amounts of mRNA for IFN-gamma were detected in Ag sites by QC-RT/PCR at 6 and 24 h, but were not significantly different than at B sites. The frequency of IFN-gamma mRNA(+)cells was higher in Ag than in B sites at 6 h. There was no correlation between the amount if IL-5 detected by QC-RT/PCR and frequency of IL-5 mRNA(+)cells by ISH. These findings also did not correlate with the degree of inflammatory responses. In conclusion: (1) greater IL-5 than IFN-gamma deposition in Ag sites suggests Th(2)predominance in LPR; (2) lack of correlation between QC-RT/PCR and ISH findings may reflect varying mRNA content of inflammatory cells.     

Tissue distribution and processing of proSAAS by proprotein convertases

The conversion of inactive precursor proteins into bioactive neuropeptides and peptide hormones involves regulated secretory proteins such as prohormone convertases PC1 and PC2. The neuroendocrine protein 7B2 represents a specific binding protein for PC2, and the protein proSAAS, which interacts with PC1, exhibits certain structural and functional homologies with 7B2. With the intention of better understanding the physiological role of proSAAS and its derived peptides, we investigated its tissue localization using a new radioimmunoassay (RIA) to a C-terminal proSAAS-derived peptide. Immunoreactivity corresponding to this SAAS-derived peptide is mostly localized to the brain and gut. Analysis of the brain distribution of the proSAAS-derived peptides indicates that the hypothalamus and pituitary are the two richest areas, consistent with the previously described high expression of PC1 in these two areas. In order to investigate the cleavage of proSAAS by prohormone convertases, we incubated recombinant His-tagged proSAAS with recombinant mouse proPC2 or furin, separated the cleavage products using high-pressure gel permeation chromatography and analyzed the products by RIA. Our results indicate that either PC2 or furin can accomplish in vitro rapid removal and efficient internal processing of the C-terminal peptide, exposing the inhibitory hexapeptide to possible further digestion by carboxypeptidases. Finally, we also studied proSAAS processing in the brains of wild-type and PC2 null mice and found that proSAAS is efficiently processed in vivo. Whereas the C-terminal peptide is mostly internally cleaved in wild-type mouse brain, it is not processed as efficiently in the brain of PC2 null mice, suggesting that PC2 is partially responsible for this cleavage in vivo.     

Cell-specific expression of CYP2A5 in the mouse respiratory tract: effects of olfactory toxicants.

We performed a detailed analysis of mouse cytochrome P450 2A5 (CYP2A5) expression by in situ hybridization (ISH) and immunohistochemistry (IHC) in the respiratory tissues of mice. The CYP2A5 mRNA and the corresponding protein co-localized at most sites and were predominantly detected in the olfactory region, with an expression in sustentacular cells, Bowman's gland, and duct cells. In the respiratory and transitional epithelium there was no or only weak expression. The nasolacrimal duct and the excretory ducts of nasal and salivary glands displayed expression, whereas no expression occurred in the acini. There was decreasing expression along the epithelial linings of the trachea and lower respiratory tract, whereas no expression occurred in the alveoli. The hepatic CYP2A5 inducers pyrazole and phenobarbital neither changed the CYP2A5 expression pattern nor damaged the olfactory mucosa. In contrast, the olfactory toxicants dichlobenil and methimazole induced characteristic changes. The damaged Bowman's glands displayed no expression, whereas the damaged epithelium expressed the enzyme. The CYP2A5 expression pattern is in accordance with previously reported localization of protein and DNA adducts and the toxicity of some CYP2A5 substrates. This suggests that CYP2A5 is an important determinant for the susceptibility of the nasal and respiratory epithelia to protoxicants and procarcinogens.     

Detection of MET mRNA in gastric cancer in situ. Comparison with immunohistochemistry and sandwich immunoassays

Determination of predictive biomarkers by immunohistochemistry (IHC) relies on antibodies with high selectivity. RNA in situ hybridization (RNA ISH) may be used to confirm IHC and may potentially replace it if suitable antibodies are not available or are insufficiently selective to discriminate closely related protein isoforms. We validated RNA ISH as specificity control for IHC and as a potential alternative method for selecting patients for treatment with MET inhibitors. MET, the HGF receptor, is encoded by the MET proto-oncogene that may be activated by mutation or amplification. MET expression and activity were tested in a panel of control cell lines. MET could be detected in formalin fixed paraffin, embedded (FFPE) samples by IHC and RNA ISH, and this was confirmed by sandwich immunoassays of fresh frozen samples. Gastric cancer cell lines with high MET expression and phosphorylation of tyrosine-1349 respond to the MET inhibitor, BAY-853474. High expression and phosphorylation of MET is a predictive biomarker for response to MET inhibitors. We then analyzed MET expression and activity in a matched set of FFPE vs. fresh frozen tumor samples consisting of 20 cases of gastric cancer. Two of 20 clinical samples investigated exhibited high MET expression with RNA ISH and IHC. Both cases were shown by sandwich immunoassays to exhibits strong functional activity. Expression levels and functional activity in these two cases were in a range that predicted response to treatment. Our findings indicate that owing to its high selectivity, RNA ISH can be used to confirm findings obtained by IHC and potentially may replace IHC for certain targets if no suitable antibodies are available. RNA ISH is a valid platform for testing predictive biomarkers for patient selection.     

Structure and expression of Xenopus prohormone convertase PC2.

The multifunctional prohormone, proopiomelanocortin (POMC), is processed in the melanotrope cells of the pituitary pars intermedia at pairs of basic amino acid residues to give a number of peptides, including alpha-melanophore-stimulating hormone (alpha-MSH). This hormone causes skin darkening in amphibians during background adaptation. Here we report the complete structure of Xenopus laevis prohormone convertase PC2, the enzyme thought to be responsible for processing of POMC to alpha-MSH. A comparative structural analysis revealed an overall amino acid sequence identity of 85-87% between Xenopus PC2 and its mammalian counterparts, with the lowest degree of identity in the signal peptide sequence (28-36%) and the region amino-terminal to the catalytic domain (59-60%). The occurrence of a second, structurally different PC2 protein reflects the expression of two Xenopus PC2 genes. The expression pattern of PC2 in the Xenopus pituitary gland of black- and white-adapted animals was found to be similar to that of POMC, namely high expression in active melanotrope cells of black animals. This observation is in line with a physiological role for PC2 in processing POMC to alpha-MSH.     

The Arg617-Arg618 cleavage site in the C-terminal domain of PC1 plays a major role in the processing and targeting of the enzyme within the regulated secretory pathway

The C-terminal domain of the prohormone convertase PC1 is involved in targeting of the enzyme to secretory granules in neuroendocrine cells and is subsequently processed in this compartment at an Arg617-Arg618 site. Three other dibasics are found in the C-terminal domain of mouse PC1. Here, we examined the role of the four dibasics in targeting PC1 to secretory granules. All 15 possible combinations of dibasic mutations were performed. Wild-type (WT) and mutant PC1 were stably expressed in neuroendocrine PC12 cells that lacked endogenous PC1. Processing, secretion and intracellular localization of PC1 and its mutants were analyzed. Leaving intact Arg617-Arg618 and mutating any combination of the three other dibasics yielded proteins that were stored and processed in secretory granules, similarly to WT PC1. Mutating Arg617-Arg618 alone or with any one of the three remaining dibasics generated proteins that were efficiently stored in secretory granules but were not processed further. Mutating Arg617-Arg618 with more than one of the remaining dibasics produced proteins that reached the TGN but were not stored in secretory granules and exited the cells through the constitutive secretory pathway. These data demonstrate that the Arg617-Arg618 plays a prominent role in targeting PC1 to secretory granules.     

Eosinophil chemotactic chemokines (eotaxin, eotaxin-2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4), and C-C chemokine receptor 3 expression in bronchial biopsies from atopic and nonatopic (Intrinsic) asthmatics

Atopic (AA) and nonatopic (NAA) asthma are characterized by chronic inflammation and local tissue eosinophilia. Many C-C chemokines are potent eosinophil chemoattractants and act predominantly via the CCR3. We examined the expression of eotaxin, eotaxin-2, RANTES, monocyte chemoattractant protein-3 (MCP-3), MCP-4, and CCR3 in the bronchial mucosa from atopic (AA) and nonatopic (intrinsic; NAA) asthmatics and compared our findings with atopic (AC) and nonatopic nonasthmatic controls (NC). Cryostat sections were processed for immunohistochemistry (IHC), in situ hybridization (ISH), and double IHC/ISH. Compared with AC and NC, the numbers of EG2+ cells and the cells expressing mRNA for eotaxin, eotaxin-2, RANTES, MCP-3, MCP-4, and CCR3 were significantly increased in AA and NAA (p < 0.01). Nonsignificant differences in these variants were observed between AA and NAA and between AC and NC. Significant correlations between the cells expressing eotaxin or CCR3 and EG2+ eosinophils in the bronchial tissue were also observed for both AA (p < 0.01) and NAA (p = 0.01). Moreover, in the total asthmatic group (AA + NAA) there was a significant inverse correlation between the expression of eotaxin and that of the histamine PC20 (p < 0.05). Sequential IHC/ISH showed that cytokeratin+ epithelial cells, CD31+ endothelial cells, and CD68+ macrophages were the major sources of eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4. There was no significantly different distribution of cells expressing mRNA for these chemokines between atopic and nonatopic asthma. These findings suggest that multiple C-C chemokines, acting at least in part via CCR3, contribute to bronchial eosinophilia in both atopic and nonatopic asthma.     

A new surface antigen (PC.2) expressed exclusively on plasma cells

Somatic cell hybridization of NS.1 nonsecretor myeloma cells with spleen cells of (DBA/2 X C57BL/6)F1 mice immunized against the myeloma MOPC 70A of BALB/c mice led to the establishment of five hybridoma clones which continuously secrete anti-MOPC 70A cytotoxic antibodies. The respective antigen detected by each of the five monoclonal antibodies is expressed both on plasmacytomas and on antibody-secreting cells as the only normal cell type. The tissue distribution of this new antigen is different from that reported for the alloantigen PC.1, and we have therefore designated it as PC.2. On the basis of immune elimination of direct and indirect plaque-forming cells, all mouse strains tested express PC.2 determinants, identifying PC.2 essentially as an autoantigen. Conventional anti-PC.1 alloantiserum contains antibodies to the PC.2 determinant, and these antibodies are distinguishable from the anti-PC.1 antibodies proper by the fact that only the latter are absorbed by liver cells. Monoclonal anti-PC.2 antibodies are not directed against MuLV-(murine leukemia virus)--associated antigens as over 20 ecotropic, several MCF (mink colony forming recombinant, and xenotropic viruses failed to react in immunofluorescence assays.