The molecular complexity of tissues and the inaccessibility of most cells within a tissue limit the discovery of key targets for tissue-specific delivery of therapeutic and imaging agents in vivo. Here, we describe a hypothesis-driven, systems biology approach to identifying a small subset of proteins induced at the tissue-blood interface that are inherently accessible to antibodies injected intravenously. We use subcellular fractionation, subtractive proteomics and bioinformatics to identify endothelial cell surface proteins exhibiting restricted tissue distribution and apparent tissue modulation. Expression profiling and gamma-scintigraphic imaging with antibodies establishes two of these proteins, aminopeptidase-P and annexin A1, as selective in vivo targets for antibodies in lungs and solid tumours, respectively. Radio-immunotherapy to annexin A1 destroys tumours and increases animal survival. This analytical strategy can map tissue- and disease-specific expression of endothelial cell surface proteins to uncover novel accessible targets useful for imaging and therapy.

Endothelial cells can function differently in vitro and in vivo; however, the degree of microenvironmental modulation in vivo remains unknown at the molecular level largely because of analytical limitations. We use multidimensional protein identification technology (MudPIT) to identify 450 proteins (with three or more spectra) in luminal endothelial cell plasma membranes isolated from rat lungs and from cultured rat lung microvascular endothelial cells. Forty-one percent of proteins expressed in vivo are not detected in vitro. Statistical analysis measuring reproducibility reveals that seven to ten MudPIT measurements are necessary to achieve > or =95% confidence of analytical completeness with current ion trap equipment. Large-scale mapping of the proteome of vascular endothelial cell surface in vivo, as demonstrated here, is advisable because distinct protein expression is apparently regulated by the tissue microenvironment that cannot yet be duplicated in standard cell culture.

the efficiency of transfection. In light of the fact that ECs are difficult to transfect, the success of this method is warranted for a wider range of cells with different origins. In fact, we have obtained similar results in temporally transfecting NIH 3T3 cells using this method (data not shown). In addition, this method of temporal transfection might have utility in reversing cellular abnormalities through genetic intervention of the temporally introduced gene.

In an effort to identify regulators of aryl hydrocarbon receptor (AHR) signaling, we have employed the yeast two-hybrid system to screen for human proteins that interact in a ligand-dependent manner with the AHR. After screening 1.4 ؋ 10 6 clones from a human B cell library, two distinct clones were identified that associated specifically with the liganded receptor. No clones were identified that interacted preferentially with the unliganded AHR. One of the ligand-dependent clones, ARA9, encodes a novel 330-amino acid protein with regions of amino acid sequence similarity to the 52-kDa FK506-binding protein known to be associated with the glucocorticoid receptor. Yeast two-hybrid experiments with ARA9 demonstrated a strong interaction with the AHR that is enhanced 11-fold in the presence of the ligand ␤-naphthoflavone. In vitro experiments using proteins generated in reticulocyte lysates confirmed this interaction and indicated that ARA9 can be co-immunoprecipitated with the AHR using antisera raised specifically for either the AHR or the 90-kDa heat shock protein. The observation that ARA9 has a high affinity for both the 90-kDa heat shock protein-associated and ligand-activated forms of the AHR suggests that ARA9 is a component of the AHR-signaling pathway in vivo.The AHR 1 is a ligand-activated transcription factor that regulates the expression of xenobiotic metabolizing enzymes in response to binding environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (1). The AHR is a member of the Per-ARNT-Sim homology domain superfamily of regulatory proteins that also includes the AHR's dimer partner, ARNT, and such proteins as HIF1␣ and Sim (reviewed in Ref. 2). Members of this family are distinguished by a region of similarity of approximately 200 amino acids termed PAS (3). In the AHR this domain is involved in dimerization with other PAScontaining proteins, ligand binding, and association with hsp90 (4 -6). Most members of this superfamily also contain a basic helix-loop-helix domain immediately N-terminal to the PAS region (2). The basic domain mediates the recognition and binding of these factors to specific DNA sequences in enhancer elements that regulate transcription of target genes (6, 7). The helix-loop-helix domain functions as a primary dimerization surface that directs interactions with appropriate dimeric partners (8, 9).Although no obvious structural relationship is apparent, the AHR and certain members of the steroid receptor superfamily exhibit similarities in their signaling mechanism (10, 11). Biochemical studies have indicated that the unliganded AHR and the GR are located in the cytosol in a complex with a dimer of the molecular chaperone hsp90 and other cellular proteins (12)(13)(14). hsp90 has been shown to be an important regulator of receptor activity. Genetic studies in yeast systems deficient in hsp90 have demonstrated an absolute requirement for hsp90 in both glucocorticoid and AHR signaling (10,11,15). Biochemical studies have correlated the association of hsp90 with an incr...

The unliganded aryl hydrocarbon receptor (AHR) is found in a complex with other proteins including the 90-kDa heat shock protein (Hsp90) and a 37-kDa protein we refer to as ARA9. We found that the three tetratricopeptide repeats found in the COOH terminus of ARA9 are necessary and sufficient for interaction with the AHR complex. Conversely, the AHR's "repressor"/Hsp90 binding domain is required for interaction with ARA9. Because ARA9 closely resembles the 52-kDa FK506-binding protein (FKBP52), found in the unliganded glucocorticoid receptor (GR) complex, we compared the binding specificities of ARA9 and FKBP52 for AHR and GR. In co-immunoprecipitation experiments, ARA9 specifically associated with AHR-Hsp90 complex but not with GRHsp90 complexes. In addition, ARA9 showed a greater capacity than FKBP52 to associate with AHR-Hsp90 complexes. The biological importance of this interaction was suggested by the observation that in a yeast expression system ARA9 expression enhanced the response of AHR to the agonist ␤-napthoflavone, decreasing the EC 50 by greater than 5-fold and increasing the maximal response 2.5-fold. In contrast, co-expression of FKBP52 had no effect on AHR signaling. In addition, although ARA9 contains a domain similar to that found in other FK506-binding proteins, ARA9 binding to 3 H-FK506 could not be detected. Finally, we have characterized the developmental and expression pattern of ARA9 in the developing mouse embryo and mapped the ARA9 locus to human chromosome 11q13.3.