Kinase insert domain receptor (KDR) extracellular immunoglobulin-like domains 4-7 contain structural features that block receptor dimerization and vascular endothelial growth factor-induced signaling.

The vascular endothelial growth factor (VEGF) receptor tyrosine kinase subtype kinase insert domain receptor (KDR) contains seven extracellular Ig-like domains, of which the three most amino-terminal contain the necessary structural features required for VEGF binding. To clarify the functional role of KDR Ig-like domains 4-7, we compared VEGF-induced signaling in human embryonic kidney and porcine aortic endothelial cells expressing native versus mutant receptor proteins in which Ig-like domains 4-7, 4-6, or 7 had been deleted. Western blotting using an anti-receptor antibody indicated equivalent expression levels for each of the recombinant proteins. As expected, VEGF treatment robustly augmented native receptor autophosphorylation. In contrast, receptor autophosphorylation, as well as downstream signaling events, were VEGF-independent for cells expressing mutant receptors. (125)I-VEGF(165) bound with equal or better affinity to mutant versus native receptor, although the number of radioligand binding sites was significantly reduced because a significant percentage of mutant, but not native, receptors were localized to the cell interior. As was the case for native KDR, (125)I-VEGF(165) binding to the mutant receptors was dependent upon cell surface heparan sulfate proteoglycans, and (125)I-VEGF(121) bound with an affinity equal to that of (125)I-VEGF(165) to the native and mutant receptors. It is concluded that KDR Ig-like domains 4-7 contain structural features that inhibit receptor signaling by a mechanism that is independent of neuropilin-1 and heparan sulfate proteoglycans. We speculate that this provides a cellular mechanism for blocking unwanted signaling events in the absence of VEGF.

NCK and PAK participate in the signaling pathway by which vascular endothelial growth factor stimulates the assembly of focal adhesions.

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is a key step in the angiogenic response and is mediated, in part, by an accelerated rate of focal adhesion complex assembly and disassembly. We investigated the signaling pathway by which VEGF regulates focal adhesion complex assembly by examining the signaling proteins involved. VEGF stimulated the tyrosine phosphorylation of the SH2 domain-containing signaling proteins NCK and CRK in human umbilical vein endothelial cells. The signaling pathways that couple the kinase insert domain-containing receptor to NCK and CRK is most likely mediated by another cellular protein, as NCK and CRK were tyrosine-phosphorylated in response to VEGF in cells expressing receptors mutated at each of several candidate SH2 domain-interacting cytosolic tyrosines. In the absence of VEGF treatment, NCK (but not CRK) associated with the p21 GTPase-activated kinase PAK. PAK catalytic activity was augmented after VEGF treatment; an association of PAK with 60- and 90-kDa tyrosine-phosphorylated proteins accompanied this. VEGF stimulated the recruitment of PAK to focal adhesions, and FAK immunoprecipitated with both NCK and PAK in VEGF-treated (but not untreated) human umbilical vein endothelial cells. Inhibition of NCK protein expression using antisense oligonucleotides led to the inhibition of both VEGF-induced focal adhesion assembly and VEGF-induced cell migration, demonstrating a necessary role of NCK in these cellular responses.

Vascular endothelial growth factor-induced migration of vascular smooth muscle cells in vitro.

Angiogenesis is a complex process that includes recruitment and proliferation of mural cells-smooth muscle cells (SMC) and pericytes. Vascular endothelial growth factor (VEGF) has been shown to play an important role in angiogenesis and is an endothelial cell chemoattractant. In addition, certain VEGF isoforms have been implicated in the normal formation of smooth muscle cell-surrounded arteries. Because VEGF's role as a mural cell chemoattractant had not been explored, we examined the ability of VEGF to influence vascular SMC migration in vitro. A Boyden chamber migration assay demonstrated that VEGF (0-100 ng/ml) caused a dose-dependent migration of SMC. VEGF did not cause proliferation of SMC. Reverse transcriptase-polymerase chain reaction analysis demonstrated the presence of both KDR and flt mRNA, two known VEGF receptors, in SMC cultures. Western blot analysis of SMC lysates confirmed these data, revealing bands migrating at approximately 200 kDa and slightly below 200 kDa consistent with KDR and flt. These observations demonstrate that VEGF receptors are present on SMC, and that VEGF can act as an SMC chemoattractant.

Angiogenesis activators and inhibitors differentially regulate caveolin-1 expression and caveolae formation in vascular endothelial cells. Angiogenesis inhibitors block vascular endothelial growth factor-induced down-regulation of caveolin-1.

Angiogenesis is the process by which new blood vessels are formed via proliferation of vascular endothelial cells. A variety of angiogenesis inhibitors that antagonize the effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have recently been identified. However, the mechanism by which these diverse angiogenesis inhibitors exert their common effects remains largely unknown. Caveolin-1 and -2 are known to be highly expressed in vascular endothelial cells both in vitro and in vivo. Here, we examine the potential role of caveolins in the angiogenic response. For this purpose, we used the well established human umbilical vein endothelial cell line, ECV 304. Treatment of ECV 304 cells with known angiogenic growth factors (VEGF, bFGF, or hepatocyte growth factor/scatter factor), resulted in a dramatic reduction in the expression of caveolin-1. This down-regulation event was selective for caveolin-1, as caveolin-2 levels remained constant under these conditions of growth factor stimulation. VEGF-induced down-regulation of caveolin-1 expression also resulted in the morphological loss of cell surface caveolae organelles as seen by transmission electron microscopy. A variety of well characterized angiogenesis inhibitors (including angiostatin, fumagillin, 2-methoxy estradiol, transforming growth factor-beta, and thalidomide) effectively blocked VEGF-induced down-regulation of caveolin-1 as seen by immunoblotting and immunofluorescence microscopy. However, treatment with angiogenesis inhibitors alone did not significantly affect the expression of caveolin-1. PD98059, a specific inhibitor of mitogen-activated protein kinase and a known angiogenesis inhibitor, also blocked the observed VEGF-induced down-regulation of caveolin-1. Furthermore, we show that caveolin-1 can function as a negative regulator of VEGF-R (KDR) signal transduction in vivo. Thus, down-regulation of caveolin-1 may be an important step along the pathway toward endothelial cell proliferation.

Autophosphorylation of KDR in the kinase domain is required for maximal VEGF-stimulated kinase activity and receptor internalization.

We have previously reported the identification of four autophosphorylation sites on the KDR VEGF receptor. Two of these sites (tyrosines 951 and 996) are located in the receptor's kinase insert domain, and two (tyrosines 1054 and 1059) are located in the catalytic domain. In order to clarify the functional significance of these sites, we made DNA constructs in which tyrosine codons were replaced with those for phenylalanine, and expressed the DNA constructs in 293 cells. VEGF binding to cells expressing the native receptor led to a rapid increase in receptor and PLC-gamma phosphorylation, and a slower increase in the phosphorylation of p125FAK and paxillin. VEGF binding to KDR(Y951F) and KDR(Y996F) expressing cells resulted in phosphorylation of all cellular substrates tested, although the level of PLCgamma phosphorylation was decreased for KDR(Y996F). The decreased level of PLCgamma phosphorylation was not because PLCgamma-containing SH2 domains bind to the Y996 autophosphorylation site. We conclude that there exists receptor autophosphorylation sites not previously identified which allow for signaling via PLCgamma, as well as p125FAK and paxillin. VEGF binding to cells expressing KDR mutated at both tyrosine's 1054 and 1059 activated receptor autophosphorylation but at a level which was only 10% of that seen for cells expressing native receptor. Tyrosine phosphorylation of cell signaling proteins was not observed in KDR(Y1054,1059) expressing cells. Utilizing an in vitro assay which directly measures receptor catalytic activity allowed us to determine that the tyrosine kinase activity of the native receptor was significantly greater than that for the double mutant. We conclude from this result that VEGF-induced autophosphorylation at tyrosines 1054 and 1059 is a required step for allowing maximal KDR kinase activity. Maximal rates of receptor kinase activity is required for VEGF-induced receptor internalization, as internalization was delayed in the KDR(Y1054,1059F) expressing cells when compared to cells expressing native receptor.

Characterization of a soluble vascular endothelial growth factor receptor-immunoglobulin chimera.

To investigate the interaction between vascular endothelial growth factor (VEGF) and its receptor, we have constructed a chimeric protein consisting of the extracellular ligand-binding domain of the human VEGF receptor subtype KDR fused to a human IgG1 Fc domain (KDR-Fc). KDR-Fc was expressed in human 293 kidney epithelial cells as a 300-kDa secreted, dimeric glycoprotein that bound 125I-VEGF165 with high affinity (Kd = 150 pM). Unlike the full length cellular receptor, KDR-Fc did not require heparin for 125I-VEGF165 binding, although heparin did stimulate 125I-VEGF165 binding approximately 50 to 100%. Similar results were observed for KDR-Fc expressed in yeast cells. Since yeast do not synthesize heparan sulfate proteoglycans, we conclude that cellular heparan sulfates do not account for the lack of a heparin requirement for 125I-VEGF165 binding to KDR-Fc. The polycationic protein protamine, which inhibits (IC50 = 1 microgram/ml) 125I-VEGF165 binding to bovine aortic endothelial cells and other KDR-expressing cells by blocking heparin interactions, had no effect on the heparin independent component of 125I-VEGF165 binding to KDR-Fc. Protamine does inhibit (IC50 = 1 microgram/ml) the heparin dependent component of 125I-VEGF165 binding to KDR-Fc. KDR-Fc bound VEGF121 with the same affinity as VEGF165. Heparin had no effect on 125I-VEGF121 binding to KDR-Fc, indicating that heparin interaction with the 44 amino acids contained in VEGF165 but not VEGF121 allow for maximal VEGF165 binding. Deletion analysis of KDR-Fc demonstrated that the determinants required for high affinity VEGF binding are located in the three aminoterminal Ig-domains of the protein. Heparin had no effect on 125I-VEGF165 binding to the three Ig-domain receptor, suggesting that there are heparin binding determinants located in KDR Ig-domains 4 to 7.

Identification of a heparin binding peptide on the extracellular domain of the KDR VEGF receptor.

Vascular endothelial growth factor (VEGF), a potent and specific activator of endothelial cells, is expressed as multiple homodimeric forms resulting from alternative RNA splicing. VEGF121 does not bind heparin while the other three isoforms do, and it has been documented that the binding of VEGF165 to its receptor is dependent upon cell surface heparin sulfate proteoglycans. Little is known about the biochemical mechanism that allows for heparin regulation of growth factor binding. For example, it is not clear whether heparin interactions with growth factor or with cell surface receptors or both are essential for VEGF binding to its receptor. In this manuscript we provide results which are consistent with the hypothesis that an interaction between heparin and a site on the KDR receptor subtype is essential for VEGF165 binding. First, we demonstrate that expression of KDR into a CHO cell line deficient in heparan sulfate biosynthesis does not allow VEGF165 binding unless heparin is exogenously added during the binding assay. Secondly, we show that a ten amino acid synthetic peptide, corresponding to a sequence from the extracellular domain of the KDR, both inhibits VEGF165 binding to the receptor and also binds heparin with high avidity. Third, affinity purification of heparin molecules on a KDR-derived peptide affinity column, together with capillary electrophoresis and polyacrylamide electrophoresis analysis, was used to show that the KDR-derived peptide interacts with a specific subset of polysaccharide chains contained in the unfractionated heparin. Taken together, these results are consistent with the hypothesis that interactions between cell surface heparan sulfate proteoglycans and the VEGF receptor contribute to allowing maximal VEGF binding.

Expression of the vascular endothelial growth factor receptor, KDR, in human placenta.

Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor known to act directly on vascular endothelial cells by promoting cell proliferation and permeability. To date, 3 structurally related cell surface receptors for VEGF, Flt-1, Flt-4 and KDR, have been identified and shown to be human type III receptor tyrosine kinases. The establishment of a vascular network is crucial to the development of the placenta and occurs through both angiogenesis and vasculogenesis. The signals controlling these processes are unclear. Immunohistochemical and in situ hybridisation techniques have localised VEGF in the trophoblast layers and VEGF binding to placental vascular endothelial cells and haemangioblasts has been shown, suggesting a role for VEGF and its receptors in development of the vascular network. In this study we have used specific antibodies to localise KDR and endothelial cells in 1st and 3rd trimester human placenta. The staining showed a colocalisation of KDR with endothelial cells and haemangioblasts. No staining of trophoblast cells was observed, but strong staining of the endothelial cells was seen in the villous stroma adjacent to areas of trophoblast proliferation.

The kinase insert domain receptor gene (KDR) has been relocated to chromosome 4q11-->q12.

Through in situ hybridization of a genomic DNA probe to metaphase chromosomes, we have localized the KDR gene to 4q11-->q12. This is the same locus as that for two other receptor tyrosine kinases, PDGFRA and KIT. This location for KDR differs from that which we previously reported using a cDNA probe. Using cDNA probes for both KDR and KIT identifies a locus at 4q31-->q32 which may uncover another cluster of receptor tyrosine kinase genes.

Biological activity and phosphorylation sites of the bacterially expressed cytosolic domain of the KDR VEGF-receptor.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor which binds to two structurally similar receptor tyrosine kinases, KDR and FLT1. Towards the goal of clarifying the signal transduction pathways by which VEGF activates endothelial cells, we expressed in bacteria an enzymatically active form of the cytosolic domain of the KDR receptor. The expressed protein undergoes autophosphorylation in both bacterial cells and in its purified form. Using peptide mapping and sequencing of peptides, we identified four tyrosine residues that are phosphorylated corresponding to residues 951, 996, 1054, and 1059 of the KDR protein. The location of the phosphorylated residues in the bacterially expressed protein, and/or the consensus sequences around these sites, suggest they may be identical to the phosphorylated sites of KDR in mammalian cells.

The related FLT4, FLT1, and KDR receptor tyrosine kinases show distinct expression patterns in human fetal endothelial cells.

The growth factor receptors expressed on endothelial cells are of special interest because of their potential to program endothelial cell growth and differentiation during development and neovascularization in various pathological states, such as wound healing and angiogenesis associated with tumorigenesis. Vascular endothelial growth factor ([VEGF] also known as vascular permeability factor) is a potent mitogen and permeability factor, which has been suggested to play a role in embryonic and tumor angiogenesis. The newly cloned FLT4 receptor tyrosine kinase gene encodes a protein related to the VEGF receptors FLT1 and KDR/FLK-1. We have here studied the expression of FLT4 and the other two members of this receptor family in human fetal tissues by Northern and in situ hybridization. These results were also compared with the sites of expression of VEGF and the related placenta growth factor (PlGF). Our results reveal FLT4 mRNA expression in vascular endothelial cells in developing vessels of several organs. A comparison of FLT4, FLT1 and KDR/FLK-1 receptor mRNA signals shows overlapping, but distinct expression patterns in the tissues studied. Certain endothelia lack one or two of the three receptor mRNAs. These data suggest that the receptor tyrosine kinases encoded by the FLT gene family may have distinct functions in the regulation of the growth/differentiation of blood vessels.

Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.

Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen which stimulates angiogenesis. Here we report that a previously identified receptor tyrosine kinase gene, KDR, encodes a receptor for VEGF. Expression of KDR in CMT-3 (cells which do not contain receptors for VEGF) allows for saturable 125I-VEGF binding with high affinity (KD = 75 pM). Affinity cross-linking of 125I-VEGF to KDR-transfected CMT-3 cells results in specific labeling of two proteins of M(r) = 195 and 235 kDa. The KDR receptor tyrosine kinase shares structural similarities with a recently reported receptor for VEGF, flt, in a manner reminiscent of the similarities between the alpha and beta forms of the PDGF receptors.

The KDR gene maps to human chromosome 4q31.2----q32, a locus which is distinct from locations for other type III growth factor receptor tyrosine kinases.

KDR (kinase insert domain receptor), a new type III receptor tyrosine kinase gene, maps to human chromosome 4q31.2----q32 by fluorescence in situ hybridization. This differs from the chromosomal locations of other members of this gene family.

Identification of a new endothelial cell growth factor receptor tyrosine kinase.

A new growth factor receptor tyrosine kinase (RTK) gene (designated KDR) has been cloned from a human endothelial cell cDNA library. The gene was identified using the polymerase chain reaction (PCR) and degenerate oligonucleotide primers complementary to conserved tyrosine kinase domains that flank the insert domain, characteristic of known type III RTKs [e.g. platelet-derived growth factor receptor (PDGF-R), colony-stimulating-1 receptor (CSF-1-R), fibroblast growth factor receptor (FGF-R) and ckit]. The DNA product from PCR was then used as a probe to isolate larger DNA segments encoding the receptor from the cDNA library. The predicted amino acid sequence contained multiple characteristics (i.e. an ATP-binding site, a membrane-spanning region, split tyrosine kinase regions) typical of a type III receptor tyrosine kinase. The KDR gene is expressed as a 7.0 kb transcript, and is localized to human chromosome 4.

Identification and structural characterization of alpha 1-adrenergic receptor subtypes.

Rat liver and brain membrane alpha 1-adrenergic receptors were purified greater than 500-fold by successive chromatographic steps using heparin-agarose, an affinity matrix constructed by coupling a novel derivative of the alpha 1-selective antagonist prazosin to Affigel-102 and wheat germ agglutinin-agarose. Several lines of evidence were obtained for the existence in brain of an alpha 1-adrenergic receptor subtype that is structurally distinct from that previously characterized in liver and other tissues using photoaffinity labeling, protein purification, and DNA cloning techniques. The alpha 1-selective ligand chlorethylclonidine (CEC) (an alkylating agent) irreversibly inactivates 100% of [3H]prazosin binding sites in partially purified preparations of rat liver. Under identical conditions, only 50% of brain receptors are irreversibly inactivated. Computer modeling of data obtained from the competition by the alpha antagonists WB4101 and phentolamine for [3H]prazosin binding to partially purified preparations of rat liver is best fit by assuming a single class of low affinity sites for both ligands. However, analysis of partially purified brain preparations indicates the presence of two binding sites with different affinities for these antagonists. Additionally, prior alkylation of brain receptors with CEC results in the loss of low affinity phentolamine and WB4101 binding sites. The CEC-insensitive site in brain, which displays high affinity for phentolamine and WB4101, is resistant to photoaffinity labeling by [125I]azidoprazosin. This is not due to a markedly lower affinity of the CEC-insensitive sites for the photoaffinity label, because competition studies with [127I]azidoprazosin revealed a single class of high affinity sites in partially purified brain samples. Photoaffinity labeling of partially purified liver and brain samples not treated with CEC results in the specific labeling of a single protein of Mr 80,000. No specifically labeled protein is observed for partially purified brain samples that had previously been incubated with CEC. Treatment of photoaffinity-labeled liver and brain receptors with N-glycanase to cleave N-linked oligosaccharides results in a single Mr 55,000 protein. Taken together, these data provide evidence for the existence of a single receptor subtype (alpha 1b) in rat liver and for two subtypes (alpha 1a and alpha 1b) in rat brain. Furthermore, the insensitivity of the alpha 1a subtype to CEC and the resistance of the alpha 1a subtype to covalent labeling by an alpha 1b-selective photoaffinity probe suggest that the primary structures of the two receptor subtypes differ, such that an amino acid(s) in the alpha 1b subtype that incorporates CEC and the photoaffinity label is lacking in the alpha 1a subtype.

[The identification and characteristics of subpopulations of alpha 1-adrenergic receptors]. / Identifikatsiia i kharakteristika subpopuliatsii al'fa 1-adrenergicheskikh retseptorov.

Rat liver and brain alpha 1-adrenergic receptors were purified 500 fold by successive chromatographic steps using heparin- and wheat germ agglutinin-agarose; an affinity matrix constructed by coupling CP85.224 (a derivative of prazosin) to affigel 102. It is shown that the existence in brain of an alpha 1-adrenergic receptor subpopulation, which is structurally distinct from that previously characterized. Chlorethylclonidine, irreversibly inactivates [3H] prazosin binding sites in partially purified membrane preparations of rat liver. Under identical conditions, only 50% of receptors are irreversibly inactivated. Computer modelling of data obtained from the competition by the alpha-antagonists, WB 4101 and phentolamine, for [3H] prazosin binding to partially purified preparations of rat liver is best fit by assuming a single low-affinity site for both ligands. However, the partially purified brain preparations indicates the presence of two affinity binding sites for these antagonists. Prior alkylation of brain receptors with chlorethylclonydyne results in the loss of the low-affinity phentolamine and WB4101 binding sites. These data provide evidence for the existence of a single receptor subpopulation (alpha 1b) in rat liver and for two subpopulations (alpha 1a and alpha 1b) in rat brain. The significance of these results in understanding the signal mechanisms which allow cellular responsiveness to alpha 1-adrenergic receptor agonists is discussed.

Basic FGF treatment of endothelial cells down-regulates the 85-KDa TGF beta receptor subtype and decreases the growth inhibitory response to TGF-beta 1.

Transforming growth factor-beta 1 (TGF beta 1) and basic fibroblast growth factor (bFGF) are known to be potent inhibitors and stimulators, respectively, of endothelial cell growth in vitro. In the present study we examined the effect of bFGF on endothelial cell growth inhibitory activity of TGF beta 1 and on the binding of (125I)-TGF beta 1 to these cells. The concentration of TGF beta 1 required for half-maximal inhibition of endothelial cell growth was increased in a dose-dependent manner by bFGF (a 20-100 fold increase at 1 ng/ml of bFGF). A 24 h-pretreatment of cells with bFGF resulted in abolition of the TGF beta 1 inhibitory effect on DNA synthesis. Moreover, the binding of (125I)-TGF beta 1 to the endothelial cell surface was decreased in a concentration-dependent and time-dependent manner after a preincubation of these cells with bFGF. Analysis of the binding parameters showed that bFGF decreased by two-fold the number of TGF beta receptors (to approximately 6000 receptors per cell). Cross-linking experiments with disuccinimidyl suberate demonstrated the presence of two TGF beta receptor subtypes, a predominant 85 kDa form and a minor 65 kDa form. Basic FGF decreased selectively the labeling of the 85 kDa TGF beta receptor subtype. These findings suggest that the growth stimulator bFGF can attenuate the cell's response to the growth inhibitor TGF beta 1.

The oligosaccharide component of alpha 1-adrenergic receptors from BC3H1 and DDT1 muscle cells. Studies with glycosidases and photoaffinity labelling of intact cells.

In this study, we clarify the structural aspects of the oligosaccharides associated with the alpha 1-adrenergic receptor in two muscle cell lines. Photoaffinity labelling of intact BC3H1 or DDT1 muscle cells with 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline ([125I]azidoprazosin) followed by SDS/polyacrylamide-gel electrophoresis (PAGE) and autoradiography revealed specifically labelled proteins of molecular mass = 87,000 and 81,000, respectively. Treatment of photoaffinity-labelled receptors in DDT1 cells with 33 u. of endoglycosidase F/ml for 24 h resulted in the loss of the 81 kDa receptor and the appearance of a 52.5 kDa protein. When lower concentrations of glycosidase or shorter incubation times were used, the 81 kDa receptor was converted to a 66 kDa protein. Treatment of the photoaffinity-labelled BC3H1 receptor with endoglycosidase F resulted in the appearance of a 50.5 kDa protein. Neither alpha-mannosidase nor endoglycosidase H had an effect on the photoaffinity labelling patterns of the receptor from the two cell types. alpha 1-Adrenergic receptors, solubilized from membranes prepared from BC3H1 and DDT1 cells, bound to wheat germ agglutinin-Sepharose and were displaced by N-acetylglucosamine. Taken together, these results indicate that alpha 1-adrenergic receptors in BC3H1 and DDT1 cells contain complex, but not high, mannose oligosaccharide chains; differences in the composition or number of chains partially accounts for the different molecular mass of the receptor in the two cell lines. The results further indicate that the oligosaccharide chains contribute substantially to the apparent molecular mass of alpha 1-adrenergic receptors, as detected by SDS/PAGE, and that the protein backbone of these receptors is likely to be approximately 50 kDa.

Use of 1-deoxymannojirimycin to show that complex oligosaccharides regulate cellular distribution of the alpha 1-adrenergic receptor glycoprotein in BC3H1 muscle cells.

We have previously shown that alpha 1-adrenergic receptors in BC3H1 muscle cells are glycoproteins containing complex but not high mannose oligosaccharides. In the present study we investigated the role of the complex sugars in functional aspects of the receptor by treating BC3H1 cells with 1-deoxymannojirimycin (dMM), which blocks conversion of high mannose oligosaccharides to complex chains. Receptors were photoaffinity labeled in intact cells with 125I-azido prazosin; drug treatment with dMM resulted in conversion of the 87-kDa receptor to 62 kDa. The 62-kDa protein was sensitive to mannosidase, indicating loss of complex sugars. Radioligand ([3H]prazosin) binding analysis carried out at 37 degrees to intact cells indicated that dMM treatment increased the affinity of the alpha 1-receptors for [3H]prazosin 2-fold and decreased the number of total cellular receptors by 15%. In order to distinguish between surface and sequestered receptors, we assessed [3H]prazosin binding to intact cells at 4 degrees using competition by the hydrophilic agonist epinephrine to define surface receptors and by nonradioactive antagonists (prazosin and phentolamine) to define total receptors. In control cells, epinephrine competed for 90% of the total receptors, whereas for dMM-treated cells this value was only 60%. In addition, dMM treatment caused a 40% reduction in epinephrine-stimulated phosphatidylinositol turnover when compared with untreated cells. The results indicate that dMM treatment reduces the number of functional alpha 1-adrenergic receptors on the cell surface while increasing the number of sequestered receptors. We conclude that complex oligosaccharides are important for cellular localization and function of alpha 1-adrenergic receptors in BC3H1 cells.