Cross talk between signaling and vitamin A transport by the retinol-binding protein receptor STRA6

The plasma membrane protein STRA6 transports vitamin A from its blood carrier retinol binding protein (RBP) into cells, and it also functions as a cytokine receptor which activates JAK/STAT signaling. We show here that, unlike other cytokine receptors, phosphorylation of STRA6 is not simply induced upon binding of its extracellular ligand. Instead, activation of the receptor is triggered by STRA6-mediated translocation of retinol from serum RBP to an intracellular acceptor, the retinol-binding protein CRBP-I. The observations also demonstrate that the movement of retinol from RBP to CRBP-I, and thus activation of STRA6, is critically linked to the intracellular metabolism of the vitamin. Furthermore, the data show that STRA6 phosphorylation is required for retinol uptake to proceed. Hence, the observations demonstrate that STRA6 orchestrates a multicomponent "machinery" that couples vitamin A homeostasis and metabolism to activation of a signaling cascade and that, in turn, STRA6 signaling regulates the cellular uptake of the vitamin. STRA6 appears to be a founding member of a new class of proteins that may be termed "cytokine signaling transporters."     

The STRA6 Receptor Is Essential for Retinol-binding Protein-induced Insulin Resistance but Not for Maintaining Vitamin A Homeostasis in Tissues Other Than the Eye

The plasma membrane protein STRA6 is thought to mediate uptake of retinol from its blood carrier retinol-binding protein (RBP) into cells and to function as a surface receptor that, upon binding of holo-RBP, activates a JAK/STAT cascade. It was suggested that STRA6 signaling underlies insulin resistance induced by elevated serum levels of RBP in obese animals. To investigate these activities in vivo, we generated and analyzed Stra6-null mice. We show that the contribution of STRA6 to retinol uptake by tissues in vivo is small and that, with the exception of the eye, ablation of Stra6 has only a modest effect on retinoid homeostasis and does not impair physiological functions that critically depend on retinoic acid in the embryo or in the adult. However, ablation of Stra6 effectively protects mice from RBP-induced suppression of insulin signaling. Thus one biological function of STRA6 in tissues other than the eye appears to be the coupling of circulating holo-RBP levels to cell signaling, in turn regulating key processes such as insulin response.     

Transthyretin Blocks Retinol Uptake and Cell Signaling by the Holo-Retinol-Binding Protein Receptor STRA6

Vitamin A is secreted from cellular stores and circulates in blood bound to retinol-binding protein (RBP). In turn, holo-RBP associates in plasma with transthyretin (TTR) to form a ternary RBP-retinol-TTR complex. It is believed that binding to TTR prevents the loss of RBP by filtration in the kidney. At target cells, holo-RBP is recognized by STRA6, a plasma membrane protein that serves a dual role: it mediates uptake of retinol from extracellular RBP into cells, and it functions as a cytokine receptor that, upon binding holo-RBP, triggers a JAK/STAT signaling cascade. We previously showed that STRA6-mediated signaling underlies the ability of RBP to induce insulin resistance. However, the role that TTR, the binding partner of holo-RBP in blood, plays in STRA6-mediated activities remained unknown. Here we show that TTR blocks the ability of holo-RBP to associate with STRA6 and thereby effectively suppresses both STRA6-mediated retinol uptake and STRA6-initiated cell signaling. Consequently, TTR protects mice from RBP-induced insulin resistance, reflected by reduced phosphorylation of insulin receptor and glucose tolerance tests. The data indicate that STRA6 functions only under circumstances where the plasma RBP level exceeds that of TTR and demonstrate that, in addition to preventing the loss of RBP, TTR plays a central role in regulating holo-RBP/STRA6 signaling.     

STRA6-Catalyzed Vitamin A Influx, Efflux, and Exchange

Vitamin A has diverse biological functions and is essential for human survival. STRA6 is the high-affinity membrane receptor for plasma retinol binding protein (RBP), the principle and specific carrier of vitamin A (retinol) in the blood. It was previously shown that STRA6 couples to lecithin retinol acyltransferase (LRAT) and cellular retinol binding protein I (CRBP-I), but poorly to CRBP-II, for retinol uptake from holo-RBP. STRA6 catalyzes both retinol release from holo-RBP, which is responsible for its retinol uptake activity, and the loading of free retinol into apo-RBP, which can cause retinol efflux. Although STRA6-catalyzed retinol efflux into apo-RBP can theoretically deplete cells of retinoid, it is unclear to what extent this efflux happens and in what context. We show here that STRA6 can couple strongly to both CRBP-I and CRBP-II for retinol efflux to apo-RBP. Strikingly, pure apo-RBP can cause almost complete depletion of retinol taken up by CRBP-I in a STRA6-dependent manner. However, if STRA6 encounters both holo-RBP and apo-RBP (as in blood), holo-RBP blocks STRA6-mediated retinol efflux by competing with apo-RBP’s binding to STRA6 and by counteracting retinol efflux with influx. We also found that STRA6 catalyzes efficient retinol exchange between intracellular CRBP-I and extracellular RBP, even in the presence of holo-RBP. STRA6’s retinol exchange activity may serve to refresh the intracellular retinoid pool. This exchange is also a previously unknown function of CRBP-I and distinguishes CRBP-I from LRAT.     

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor''s vitamin A uptake mechanism.     

Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor

STRA6 is a multitransmembrane domain protein not homologous to any other proteins with known function. It functions as the high-affinity receptor for plasma retinol binding protein (RBP) and mediates cellular uptake of vitamin A from the vitamin A-RBP complex. Consistent with the diverse roles of vitamin A and the wide tissue expression pattern of STRA6, mutations in STRA6 are associated with severe pathological phenotypes in humans. The structural basis for STRA6's biochemical function is unknown. Although computer programs predict 11 transmembrane domains for STRA6, its topology has never been studied experimentally. Elucidating the transmembrane topology of STRA6 is critical for understanding its structure and function. By inserting an epitope tag into all possible extracellular and intracellular domains of STRA6, we systematically analyzed the accessibility of each tag on the surface of live cells, the accessibility of each tag in permeabilized cells, and the effect of each tag on RBP binding and STRA6-mediated vitamin A uptake from the vitamin A-RBP complex. In addition, we used a new lysine accessibility technique combining cell-surface biotinylation and tandem-affinity purification to study a region of the protein not revealed by the epitope tagging method. These studies not only revealed STRA6's extracellular, transmembrane, and intracellular domains but also implicated extracellular regions of STRA6 in RBP binding.     

Differential and Isomer-Specific Modulation of Vitamin A Transport and the Catalytic Activities of the RBP Receptor by Retinoids

Retinoids are vitamin A derivatives with diverse biological functions. Both natural and artificial retinoids have been used as therapeutic reagents to treat human diseases, but not all retinoid actions are understood mechanistically. Plasma retinol binding protein (RBP) is the principal and specific carrier of vitamin A in the blood. STRA6 is the membrane receptor for RBP that mediates cellular vitamin A uptake. The effects of retinoids or related compounds on the receptor’s vitamin A uptake activity and its catalytic activities are not well understood. In this study, we dissected the membrane receptor-mediated vitamin A uptake mechanism using various retinoids. We show that a subset of retinoids strongly stimulates STRA6-mediated vitamin A release from holo-RBP. STRA6 also catalyzes the exchange of retinol in RBP with certain retinoids. The effect of retinoids on STRA6 is highly isomer-specific. This study provides unique insights into the RBP receptor’s mechanism and reveals that the vitamin A transport machinery can be a target of retinoid-based drugs.     

Retinol-binding protein 4 downregulation during osteogenesis and its localization to non-endocytic vesicles in human cranial suture mesenchymal cells suggest a novel tissue function

Craniosynostosis is a developmental disorder of the skull arising from premature bony fusion of cranial sutures, the sites of skull bone growth. In a recent gene microarray study, we demonstrated that retinol-binding protein 4 (RBP4) was the most highly downregulated gene in suture tissue during the pathological process of premature bony fusion. To gain insight into the function of RBP4 in cranial sutures, we analysed primary cells cultured from human cranial suture mesenchyme. These cells express RBP4 but not CRBP1, cellular retinol-binding protein 1, the typical cytoplasmic retinol storage protein. Using flow cytometry, we showed that suture mesenchymal cells express the RBP4 receptor, STRA6, on the cell surface. In a cell culture model of cranial osteogenesis, we found that RBP4 was significantly downregulated during mineralization, analogous to its decrease in pathological suture fusion. We found that cranial suture cells do not secrete detectable levels of RBP4, suggesting that it acts in a cell-autonomous manner. High-resolution confocal microscopy with a panel of antibody markers of cytoplasmic organelles demonstrated that RBP4 was present in several hundred cytoplasmic vesicles of about 300 nm in diameter which, in large part, were conspicuously distinct from the ER, the Golgi and endosomes of the endocytic pathway. We speculate that in suture mesenchymal cells, endogenous RBP4 receives retinol from STRA6 and the RBP4-retinol complex is stored in vesicles until needed for conversion to retinoic acid in the process of osteogenesis. This study extends the role of RBP4 beyond that of a serum transporter of retinol and implicates a broader role in osteogenesis.     

An essential ligand-binding domain in the membrane receptor for retinol-binding protein revealed by large-scale mutagenesis and a human polymorphism

Plasma retinol-binding protein (RBP), the principal carrier of vitamin A in the blood, delivers vitamin A from liver, the site of storage, to distant organs that need vitamin A, such as the eye, brain, placenta, and testis. STRA6 is a high-affinity membrane receptor for RBP and mediates vitamin A uptake in these target organs. STRA6 is a 74-kDa multi-transmembrane domain protein that represents a new class of membrane transport protein. In this study, we used an unbiased strategy by analyzing >900 random mutants of STRA6 to study its structure and function, and we identified an essential RBP-binding domain in STRA6. Mutations in any of the three essential residues in this domain can almost completely abolish binding of STRA6 to RBP and its vitamin A uptake activity from holo-RBP without affecting its cell surface expression. We have also functionally characterized the mutations in human STRA6 that cause severe birth defects as well as several human polymorphisms. All STRA6 mutants associated with severe birth defects have largely abolished vitamin A uptake activity, consistent with the severe clinical phenotypes. In addition, we have identified a human polymorphism that significantly reduces the vitamin A uptake activity of STRA6. Interestingly, the residue affected by this polymorphism is located in the RBP-binding domain we identified, and the polymorphism causes decreased vitamin A uptake by reducing RBP binding. This study identifies an essential functional domain in STRA6 and a human polymorphism in this domain that leads to reduced vitamin A uptake activity.     

Retinol-Binding Protein 4 and Its Membrane Receptor STRA6 Control Adipogenesis by Regulating Cellular Retinoid Homeostasis and Retinoic Acid Receptor α Activity

Retinoids are vitamin A (retinol) derivatives and complex regulators of adipogenesis by activating specific nuclear receptors, including the retinoic acid receptor (RAR) and retinoid X receptor (RXR). Circulating retinol-binding protein 4 (RBP4) and its membrane receptor STRA6 coordinate cellular retinol uptake. It is unknown whether retinol levels and the activity of RAR and RXR in adipocyte precursors are linked via RBP4/STRA6. Here, we show that STRA6 is expressed in precursor cells and, dictated by the apo- and holo-RBP4 isoforms, mediates bidirectional retinol transport that controls RARα activity and subsequent adipocyte differentiation. Mobilization of retinoid stores in mice by inducing RBP4 secretion from the liver activated RARα signaling in the precursor cell containing the stromal-vascular fraction of adipose tissue. Retinol-loaded holo-RBP4 blocked adipocyte differentiation of cultured precursors by activating RARα. Remarkably, retinol-free apo-RBP4 triggered retinol efflux that reduced cellular retinoids, RARα activity, and target gene expression and enhanced adipogenesis synergistically with ectopic STRA6. Thus, STRA6 in adipocyte precursor cells links nuclear RARα activity to the circulating RBP4 isoforms, whose ratio in obese mice was shifted toward limiting the adipogenic potential of their precursors. This novel cross talk identifies a retinol-dependent metabolic function of RBP4 that may have important implications for the treatment of obesity.     

O-GlcNAcylation disrupts STRA6-retinol signals in kidneys of diabetes

BackgroundO-GlcNAcylation is an important mechanism of diabetic complication. Retinoid homeostasis regulates cell-physiological functions through STRA6-retinol signaling. Therefore, we investigated whether O-GlcNAcylation disrupted STRA6-retinol signals in diabetes.MethodsImmunoprecipitation and proximity ligation assay were used to investigate O-GlcNAcylation of STRA6-retinol signals in kidneys of db/db and ob/ob mice. Western blot and immunohistochemistry were done for STRA6/CRBP1/LRAT/RALDH1/RARs pathway, GFAT, OGT, TGFβ₁ and collagen 1 level. HPLC and ELISA for retinol, retinal, and retinoic acid concentrations were performed in vivo and vitro. RBP4 binding with STRA6 was measured in vitro. To verify whether O-GlcNAcylation disrupted STRA6-retinol signals, treatment of TMG and OSMI-1, transfection of OGA and OGT, and OGT siRNA were performed in HK-2 cells.ResultsSTRA6 and RALDH1 were highly O-GlcNAc-modified in glomeruli and tubules of db/db and ob/ob mice. RBP4, p-Try, p-JAK2, and p-STAT5 on STRA6 immunoprecipitate were reduced. Cellular retinol signals (CRBP1, LRAT, ADH, retinol, retinal, RA, RARα, RARγ and RXRα) remarkably decreased in kidneys of db/db, ob/ob mice and HG-cultured cells. TMG and OGT transfection induced O-GlcNAcylation of STRA6 and RALDH1, repressed RBP4-bound STRA6, and retinol signals in NG-cultured cells. OSMI-1, OGA transfection, and OGT silence reversed O-GlcNAc-modification of STRA6 and RALDH1, and rescued the decrease of retinol signals, and reversed the increase of TGFβ₁ and collagen 1 in HG-treated cells.ConclusionsO-GlcNAcylation significantly modified STRA6 and RALDH1, suppressed RBP4 binding activity, and disrupted retinol signals in the kidney of diabetes.General significanceThis study first indicates that STRA6-retinol signals were directly disrupted by O-GlcNAcylation in diabetic kidney.     

Vitamin A Transport and the Transmembrane Pore in the Cell-Surface Receptor for Plasma Retinol Binding Protein

Vitamin A and its derivatives (retinoids) play diverse and crucial functions from embryogenesis to adulthood and are used as therapeutic agents in human medicine for eye and skin diseases, infections and cancer. Plasma retinol binding protein (RBP) is the principal and specific vitamin A carrier in the blood and binds vitamin A at 1∶1 ratio. STRA6 is the high-affinity membrane receptor for RBP and mediates cellular vitamin A uptake. STRA6 null mice have severely depleted vitamin A reserves for vision and consequently have vision loss, even under vitamin A sufficient conditions. STRA6 null humans have a wide range of severe pathological phenotypes in many organs including the eye, brain, heart and lung. Known membrane transport mechanisms involve transmembrane pores that regulate the transport of the substrate (e.g., the gating of ion channels). STRA6 represents a new type of membrane receptor. How this receptor interacts with its transport substrate vitamin A and the functions of its nine transmembrane domains are still completely unknown. These questions are critical to understanding the molecular basis of STRA6′s activities and its regulation. We employ acute chemical modification to introduce chemical side chains to STRA6 in a site-specific manner. We found that modifications with specific chemicals at specific positions in or near the transmembrane domains of this receptor can almost completely suppress its vitamin A transport activity. These experiments provide the first evidence for the existence of a transmembrane pore, analogous to the pore of ion channels, for this new type of cell-surface receptor.     

Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

Serum retinol-binding protein 4 (RBP4) is the sole specific vitamin A (retinol) transporter in blood. Elevation of serum RBP4 in patients has been linked to cardiovascular disease and diabetic retinopathy. However, the significance of RBP4 elevation in the pathogenesis of these vascular diseases is unknown. Here we show that RBP4 induces inflammation in primary human retinal capillary endothelial cells (HRCEC) and human umbilical vein endothelial cells (HUVEC) by stimulating expression of proinflammatory molecules involved in leukocyte recruitment and adherence to endothelium, including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin, monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). We demonstrate that these novel effects of RBP4 are independent of retinol and the RBP4 membrane receptor STRA6 and occur in part via activation of NADPH oxidase and NF-κB. Importantly, retinol-free RBP4 (apo-RBP4) was as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory molecules in both HRCEC and HUVEC. These studies reveal that RBP4 elevation can directly contribute to endothelial inflammation and therefore may play a causative role in the development or progression of vascular inflammation during cardiovascular disease and microvascular complications of diabetes.     

RBP4 disrupts vitamin A uptake homeostasis in a STRA6-deficient animal model for Matthew-Wood syndrome

The cellular uptake of vitamin A from its RBP4-bound circulating form (holo-RBP4) is a homeostatic process that evidently depends on the multidomain membrane protein STRA6. In humans, mutations in STRA6 are associated with Matthew-Wood syndrome, manifested by multisystem developmental malformations. Here we addressed the metabolic basis of this inherited disease. STRA6-dependent transfer of retinol from RBP4 into cultured NIH 3T3 fibroblasts was enhanced by lecithin:retinol acyltransferase (LRAT). The retinol transfer was bidirectional, strongly suggesting that STRA6 acts as a retinol channel/transporter. Loss-of-function analysis in zebrafish embryos revealed that Stra6 deficiency caused vitamin A deprivation of the developing eyes. We provide evidence that, in the absence of Stra6, holo-Rbp4 provokes nonspecific vitamin A excess in several embryonic tissues, impairing retinoic acid receptor signaling and gene regulation. These fatal consequences of Stra6 deficiency, including craniofacial and cardiac defects and microphthalmia, were largely alleviated by reducing embryonic Rbp4 levels by morpholino oligonucleotide or pharmacological treatments.     

High-resolution structures of retinol-binding protein in complex with retinol: pH-induced protein structural changes in the crystal state

The targeted delivery of non-polar ligands by binding proteins to membranes or membrane receptors involves the release of these ligands on or near the plasma membrane of target cells. Because these hydrophobic ligands are often bound inside a deep cavity of binding proteins, as shown previously for plasma retinol-binding protein (RBP), their release from these proteins might require the destabilization of the protein structure by partially denaturing conditions, such as those possibly present near plasma membranes. RBP is a plasma transport protein which delivers specifically retinol from its store sites to target cells. Here, we report the high-resolution (1.1-1.4A) crystal structures of bovine holo-RBP at five different pH values, ranging from 9 to 2. While unraveling details of the native protein structure and of the interactions with retinol at nearly atomic resolution at neutral pH, this study provides evidence for definite pH-induced modifications of several structural features of RBP. The structure most representative of the changes that holo-RBP undergoes at different pH values is that of its flexible state at pH 2. At this pH, most significant are the alteration of the arrangement of salt bridges and of the network of water molecules/H-bonds that participates in the retinol-RBP interaction, an appreciable increase of the volume of the beta-barrel cavity, a considerably higher degree of mobility of the RBP-bound ligand and of several protein regions and the disorder of a large number of solvent molecules that are ordered at neutral pH. These changes are likely to be accompanied by a modification of the pattern of charge distribution on the protein surface. All these changes, which reveal a substantially lowered conformational stability of RBP, presumably occur at the initial stages of the acidic denaturation of RBP and are possibly associated with a facilitated release of the retinol molecule from its carrier protein.     

Downregulation of STRA6 in Adipocytes and Adipose Stromovascular Fraction in Obesity and Effects of Adipocyte-Specific STRA6 Knockdown In Vivo

To investigate the mechanisms by which elevated retinol-binding protein 4 (RBP4) causes insulin resistance, we studied the role of the high-affinity receptor for RBP4, STRA6 (stimulated by retinoic acid), in insulin resistance and obesity. In high-fat-diet-fed and ob/ob mice, STRA6 expression was decreased 70 to 95% in perigonadal adipocytes and both perigonadal and subcutaneous adipose stromovascular cells. To determine whether downregulation of STRA6 in adipocytes contributes to insulin resistance, we generated adipose-Stra6^−/− mice. Adipose-Stra6^−/− mice fed chow had decreased body weight, fat mass, leptin levels, insulin levels, and adipocyte number and increased expression of brown fat-selective markers in white adipose tissue. When fed a high-fat diet, these mice had a mild improvement in insulin sensitivity at an age when adiposity was unchanged. STRA6 has been implicated in retinol uptake, but retinol uptake and the expression of retinoid homeostatic genes (encoding retinoic acid receptor β [RARβ], CYP26A1, and lecithin retinol acyltransferase) were not altered in adipocytes from adipose-Stra6^−/− mice, indicating that retinoid homeostasis was maintained with STRA6 knockdown. Thus, STRA6 reduction in adipocytes in adipose-Stra6^−/− mice fed chow resulted in leanness, which may contribute to their increased insulin sensitivity. However, in wild-type mice with high-fat-diet-induced obesity and in ob/ob mice, the marked downregulation of STRA6 in adipocytes and adipose stromovascular cells does not compensate for obesity-associated insulin resistance.     

Delineation of in vivo assembled multiprotein complexes via biomolecular interaction analysis mass spectrometry

Biomolecular interaction analysis mass spectrometry (BIA-MS) is a multiplexed bioanalytical approach used in analysis of proteins from complex biological mixtures. It utilizes surface-immobilized ligands for protein affinity retrieval, surface plasmon resonance for monitoring the ligand-protein interaction and matrix-assisted laser desorption/ionization-time of flight mass spectrometry for revealing the masses of the biomolecules retrieved by the ligand. In order to explore the utility of BIA-MS in delineation of multiprotein complexes, an in vivo assembled protein complex comprised of retinol binding protein (RBP) and transthyretin (TTR) was investigated. Antibodies to RBP and TTR were utilized as ligands in the analysis of the protein complex present in human plasma. The RBP-TTR complex was retrieved by the anti-RBP antibody as indicated by the presence of both RBP and TTR signals in the mass spectra. RBP signals were not observed in the mass spectra of the material retained on the anti-TTR derivatized surface. In addition, the mass-specific detection in BIA-MS allowed detection of RBP and TTR analyte variants.     

Interactions of retinol with binding proteins: implications for the mechanism of uptake by cells

The kinetic parameters of the interaction of retinol with retinol binding protein (RBP) were studied. The rate constant for association of retinol with the protein (ka) was found to be 1.5 X 10(6) M-1 min-1. The rate constant for dissociation (kd) from the protein was determined by studying the transfer of retinol from RBP to lipid bilayers. It was found that such transfer proceeds via the aqueous phase and its rate-limiting step is the dissociation of retinol from the binding protein. The rate of transfer therefore represents the rate of dissociation. The kd was 0.112 min-1. These values were validated further by the following consideration. The equilibrium dissociation constant of RBP and retinol can be calculated from the expression Kd = kd/ka. The calculated value was 7.5 X 10(-8) M. Kd was also measured directly by fluorometric titration and was found to be 7 X 10(-8) M. The relative avidities of retinol for RBP, the complex RBP-transthyretin (RBP-TTR), and serum albumin were also studied. It was found that binding of RBP to TTR increased its avidity for retinol by about 2-fold. The avidity of albumin for retinol was 30-fold lower than that of RBP. The data imply that retinol spontaneously and rapidly dissociates from sites on binding proteins, which indicates that the vitamin can freely move in vivo between physiologic compartments with avidities for it.