We used a phage expression library of cDNAs from metastatic breast carcinoma to identify protein domains that bind to the vasculature of the lung, a frequent site of breast cancer metastasis. We found that one protein domain selectively targeted phage as well as cells to the lung. This domain is part of the protein metadherin, shown by gene expression profiling to be overexpressed in metastatic breast cancer. Immunostaining revealed that metadherin is overexpressed in breast cancer tissue and breast tumor xenografts. Antibodies reactive to the lung-homing domain of metadherin and siRNA-mediated knockdown of metadherin expression in breast cancer cells inhibited experimental lung metastasis, indicating that tumor cell metadherin mediates localization at the metastatic site.

During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca 2؉ homeostasis and the physiological roles of CCE.The two primary second messengers mediating rapid responses of cells to hormones, autacoids, and neurotransmitters are cyclic nucleotides and Ca 2ϩ . Cyclic nucleotides act, for the most part, by activating protein kinases. The actions of Ca 2ϩ are more complex, in that this cation acts in two ways: directly, by binding to effector proteins, and indirectly, by first binding to regulatory proteins such as calmodulin, troponin C, and recoverin, which in turn associate and modulate effector proteins. Effector proteins regulated in these manners by Ca 2ϩ include not only protein kinases and protein phosphatases but also phospholipases and adenylyl cyclases, which are signaling enzymes in their own right, and an array of proteins involved in cellular responses that range from muscle contraction to glycogenolysis, endo-, exo-, and neurosecretion, cell differentiation, and programmed cell death. A common mechanism used by hormones and growth factors to signal through cytosolic Ca 2ϩ ([Ca 2ϩ ] i ) is activation of a rather complex reaction cascade that begins with stimulation of phosphoinositide-specific phospholipase C (PLC) enzymes, PLC␤ and PLC␥, and is followed sequentially by formation of diacylglycerol plus inositol 1,4,5-trisphosphate (IP3), liberation of Ca 2ϩ from intracellular stores, and finally, entry of Ca 2ϩ from the external milieu. The basic mechanisms used to signal through [Ca 2ϩ ] i are determined by the fact that the resting level of cytosolic Ca 2ϩ is very low, in the neighborhood of 100 nM, while that in intracellular stores and in the surrounding extracellular milieu is in the neighborhood of 2 mM, that is, Ϸ10,000-fold higher. As a result, [Ca 2ϩ ] i is set by the balance of two opposing forces. One is passive influx into the cytoplasm. It is driven by the electrochemical gradient and causes cytosolic [Ca 2ϩ ] i to rise without expenditure of energy. This influx is carefully controlled both at the level of the plasma membrane and at the level of the membranes, which delimit the internal storage compartment. Entry of Ca 2ϩ from the extracellular space occurs through three classes of Ca 2ϩ permeable gates: voltagedependent Ca 2ϩ...

Homologues ofS timulation of cells that elevates inositol 1,4,5-trisphosphate (IP3) causes the release of Ca 2ϩ from internal stores and its entry from the external milieu (for reviews see refs. 1-3). The release from internal stores occurs through channels formed by IP3 receptors (IP3Rs), and entry is mediated by a set of functionally heterogeneous but ubiquitous channels that are activated by the store depletion event per se. Transient receptor potential (TRP) proteins have been hypothesized to be structural components of Ca 2ϩ entry channels (4, 5) and to be activated by IP3R in response to IP3 or store depletion. However, neither has the presence of TRP in Ca 2ϩ entry channels been proven nor has the mechanism(s) by which the channels are activated been clearly elucidated. Indeed, the mechanism by which Ca 2ϩ entry channels are activated has received considerable attention, and arguments have been set forth (i) for activation by second messengers or mediators that include cGMP, IP3, diacylglycerol, a G protein, arachidonic acid derivatives, and a complex termed CIF (6-14), (ii) for translocation from internal pools with involvement of an exocytotic event (15, 16), and (iii) for shortrange physical coupling between the membrane delimiting the store and the plasma membrane (17, 18). The short-range physical-coupling model proposed that membrane Ca 2ϩ entry channels may be activated by the same protein that is responsible for store depletion, i.e., the IP3R (for details see ref. 1).The first functional evidence for a direct role of IP3R in Ca 2ϩ entry was obtained by Kiselyov et al. (19), who showed that Ca 2ϩ entry channels found in HEK cells expressing transfected TRP3 in stable form can be activated in inside-out membrane patches by addition of either IP3R-rich cerebellar microsomes or liposomes carrying recombinant IP3R protein truncated at its C terminus to inactivate its channel-forming capacity. However, this study did not determine whether the protein with which IP3R interacted was TRP3. Indeed, TRP was shown to cause changes in protein expression other than TRP, e.g., upregulation of IP3Rs, and attempts to coimmunoprecipitate IP3R and TRP3 failed (19). We now show that IP3R and TRP can be coimmunoprecipitated. We thus sought to identify interacting domains using in vitro protein:protein interaction tests and, if we found them, to test for their function. Such domains were identified and, upon expression in cells whose TRP and IP3R complement had not been manipulated, were found to modulate natural Ca 2ϩ entry stimulated by either a G protein-coupled pathway or store depletion. The data support a model in which store depletion-activated Ca 2ϩ entry is mediated by TRP-based channels that are activated by the IP3R. Materials and Methods

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Capacitative Ca 2؉ entry (CCE) is Ca 2؉ entering after stimulation of inositol 1,4,5-trisphosphate (IP3) formation and initiation of Ca 2؉ store depletion. One hallmark of CCE is that it can also be triggered merely by store depletion, as occurs after inhibition of internal Ca 2؉ pumps with thapsigargin. Evidence has accumulated in support of a role of transient receptor potential (Trp) proteins as structural subunits of a class of Ca 2؉ -permeable cation channels activated by agonists that stimulate IP3 formation-very likely through a direct interaction between the IP3 receptor and a Trp subunit of the Ca 2؉ entry channel. The role of Trp's in Ca 2؉ entry triggered by store depletion alone is less clear. Only a few of the cloned Trp's appear to enhance this type of Ca 2؉ entry, and when they do, the effect requires special conditions to be observed, which native CCE does not. Here we report the full-length cDNA of mouse trp2, the homologue of the human trp2 pseudogene. Mouse Trp2 is shown to be readily activated not only after stimulation with an agonist but also by store depletion in the absence of an agonist. In contrast to other Trp proteins, Trp2-mediated Ca 2؉ entry activated by store depletion is seen under the same conditions that reveal endogenous store depletion-activated Ca 2؉ entry, i.e., classical CCE. The findings support the general hypothesis that Trp proteins are subunits of store-and receptor-operated Ca 2؉ channels.

Screening of a phage library for peptides that bind to clotted plasma in the presence of liquid plasma yielded two cyclic decapeptides, CGLIIQKNEC (CLT1) and CNAGESSKNC (CLT2). When injected intravenously into mice bearing various types of tumors, fluorescein-conjugated CLT peptides accumulated in a fibrillar meshwork in the extracellular compartment of the tumors, but were not detectable in other tissues of the tumor-bearing mice. The tumor homing of both peptides was strongly reduced after coinjection with unlabeled CLT2, indicating that the two peptides recognize the same binding site. The CLT peptide fluorescence colocalized with staining for fibrin(ogen) present in the extravascular compartment of tumors, but not in other tissues. The CLT peptides did not home to tumors grown in fibrinogen-null mice or in mice that lack plasma fibronectin. The CLT peptides also accumulated at the sites of injury in arteries, skeletal muscle, and skin. We conclude that the CLT peptides recognize fibrin-fibronectin complexes formed by clotting of plasma proteins that have leaked into the extravascular space in tumors and other lesions. These peptides may be useful in targeting diagnostic and therapeutic materials into tumors and injured tissues.fibronectin ͉ imaging ͉ phage display ͉ tumor targeting ͉ fibrin

Transient receptor potential (Trp) proteins form ion channels implicated in the calcium entry observed after stimulation of the phospholipase C pathway. KyteDoolittle analysis of the amino acid sequence of Trp proteins identifies seven hydrophobic regions (H1-H7) with potential of forming transmembrane segments. A limited sequence similarity to voltage-gated calcium channel ␣1 subunits lead to the prediction of six transmembrane (TM) segments flanked by intracellular N and C termini and a putative pore region between TM5 and TM6. However, experimental evidence supporting this model is missing. Using human Trp 3 to test Trp topology, we now confirm the intracellular nature of the termini by immunocytochemistry. We also demonstrate presence of a unique glycosylation site in position 418, which defines one extracellular loop between H2 and H3. After removal of this site and insertion of ten separate glycosylation sites, we defined two additional extracellular loops between H4 and H5, and H6 and H7. This demonstrated the existence of six transmembrane segments formed of H2-H7. Thus, the first hydrophobic region of Trp rather than being a transmembrane segment is intracellular and available for protein-protein interactions. A site placed in the center of the putative pore region was glycosylated, suggesting that this region may have been luminal and was reinserted into the membrane at a late stage of channel assembly.