Upregulation of integrins alpha v beta 3 and alpha v beta 5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery

Entry of human adenovirus into host cells involves interaction of virus particles with two distinct receptors. The initial binding event is mediated by the fiber protein, while subsequent interaction of the penton base protein with alpha v integrins promotes virus internalization and/or penetration. Although these interactions in epithelial and endothelial cells have been well characterized, relatively little is known as to whether these events occur during virus infection of human peripheral blood mononuclear cells. We demonstrate that freshly isolated peripheral blood monocytes and T lymphocytes express very small amounts of alpha v integrins and also are resistant to adenovirus infection. Exposure of monocytes to hematopoietic growth factors granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor induced expression of cell surface alpha v integrins, promoted the binding of penton base protein, and also rendered these cells susceptible to adenovirus-mediated gene delivery. Stimulation of T cells with a mitogen, phytohemagglutinin, or a cell-activating agent, phorbol myristate acetate, induced expression of alpha v integrins and also enhanced adenovirus-mediated gene delivery. These studies further indicate that alpha v integrins play a crucial role in adenovirus infection and also provide a useful strategy for enhancing adenovirus-mediated gene delivery into human peripheral blood mononuclear cells.     

Migration properties of the human ovarian adenocarcinoma cell line IGROV1: importance of alpha(v)beta3 integrins and vitronectin

Cell migration of ovarian tumoral cells is essential for cell dissemination and for invasion of the submesothelial extracellular matrix (ECM). We have conducted a study of the migratory properties of an ovarian adenocarcinoma cell line (IGROV1) by using 2 distinct methods for the evaluation of cell migration. We found that in a short-term transfilter migration assay, IGROV1 cells migrated toward vitronectin, fibronectin, type IV collagen and laminin in an integrin-dependent manner. When migration was evaluated in a wound healing assay, the restitution of the wounded area was stimulated solely by added, exogenous vitronectin and was almost totally dependent on alpha(v)beta3 integrin function. Moreover, we demonstrated that alpha(v)beta3 was localized in focal contacts restricted to the leading edge of migrating cells, whereas vitronectin notably localized with actin stress fibers and cortical actin. On the other hand, several kinase inhibitors were found to impede migration of IGROV1 induced by vitronectin. It thus appears that alpha(v)beta3-vitronectin interactions lead to the activation of multiple signaling pathway including activation of protein kinase C, phosphatidyl-inositol-3-phosphate kinase and protein tyrosine kinase. The "alpha(v)beta3-vitronectin system" is therefore essential to the migration of human ovarian carcinoma cells.     

Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors-the alpha1beta1 and alpha2beta1 integrins-through induction of mRNAs encoding the alpha1 and alpha2 subunits. In contrast, VEGF did not induce increased expression of the alpha3beta1 integrin, which also has been implicated in collagen binding. Integrin alpha1-blocking and alpha2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and alpha1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of alpha1beta1 and alpha2beta1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of alpha1-blocking and alpha2-blocking Abs. In vivo, a combination of alpha1-blocking and alpha2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of alpha1beta1 and alpha2beta1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that alpha1beta1 and alpha2beta1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

Integrins alpha(v)beta3 and alpha5beta1 mediate attachment of lyme disease spirochetes to human cells

Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin alpha(IIb)beta3 was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins alpha(v)beta3 and alpha5beta1, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified alpha(v)beta3 and alpha5beta1. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified alpha(v)beta3 and alpha5beta1 was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to alpha(v)beta3 was also shown by using a transfected cell line that expresses this receptor but not alpha(IIb)beta3. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both alpha5beta1 and alpha(v)beta3. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.     

Bone sialoprotein supports breast cancer cell adhesion proliferation and migration through differential usage of the alpha(v)beta3 and alpha(v)beta5 integrins

A high concentration of oxidized low density lipoprotein (Ox-LDL) showed a cytotoxic effect on mouse macrophage-derived J774 cells. Mutant cells were selected from these cells that were resistant to the cytotoxic effect of Ox-LDL. One mutant form, named JO21b cells, was characterized in the present study. In spite of a marked resistance to the cytotoxic effect of Ox-LDL, JO21b cells were apparently as sensitive as the parent cells not only to toxic moieties of Ox-LDL, such as 7-ketocholesterol and lysophosphatidylcholine, but also to t-butyl hydroperoxide, an artificial lipid hydroperoxide analog. However, the cellular association of 125I-labeled Ox-LDL with, and subsequent endocytic degradation by JO21b cells was reduced by 70-80% compared with J774 cells. Similarly, accumulation of cholesteryl esters in JO21b cell by Ox-LDL was also reduced by 70%. Northern blot analyses of type I and type II macrophage scavenger receptors (type I and type II MSR) demonstrated that the mRNA levels of JO21b cells were lower than those of J774 cells. Moreover, peritoneal macrophages obtained from MSR-knockout mice showed a higher resistance to the cytotoxic effect of Ox-LDL than those from their wild-type littermates. Our results suggest, therefore, that macrophage scavenger receptor-mediated endocytic uptake of oxidized low density lipoproteins (Ox-LDL) may play an enhancing role in Ox-LDL cytotoxicity to macrophages or macrophage-derived cells.     

Studies in vitro on the role of alpha v and beta 1 integrins in the adhesion of human dermal fibroblasts to provisional matrix proteins fibronectin, vitronectin, and fibrinogen

Fibroblasts that migrate into a wound during the early stages of repair use cell surface integrins to interact with extracellular molecules as they move away from the interstitial matrix of normal tissue and into the provisional matrix of the wound. Therefore, to understand a critical phase of wound healing, it is necessary to understand the details of integrin involvement. Normal adult human dermal fibroblasts in culture express many receptors for the provisional matrix proteins fibronectin, vitronectin, and fibrinogen, including the integrins alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, alpha v beta 1, alpha v beta 3, and alpha v beta 5. We used quantitative flow cytometry to estimate the relative numbers of these receptors and immunoprecipitation to confirm the expression of alpha v beta 1. Adult human dermal fibroblasts primarily use beta 1 integrins, alpha 4 beta 1, alpha 5 beta 1, and possibly alpha v beta 1, for attachment to fibronectin. alpha v beta 3 and perhaps other integrins containing the alpha v subunit serve fibroblasts as secondary or auxiliary receptors for fibronectin. In contrast, these cells use alpha v integrins but probably not beta 1 integrins for attachment to vitronectin. alpha v beta 3 and alpha v beta 5 apparently act in concert to mediate attachment to vitronectin, and these two integrins may perform different functions during wound repair. Fibroblast adhesion to certain preparations of fibrinogen occurs, at least partially, through the small amount of fibronectin present in the preparations. Fibroblast attachment to fibrinogen purified free of fibronectin also occurs, and that was demonstrated with a sensitive new assay called electrical cell-substrate impedance sensing. Fibroblast attachment to pure fibrinogen can be inhibited by RGD peptide, suggesting that integrins are involved.     

Na,K-ATPase subunit isoforms in human reticulocytes: evidence from reverse transcription-PCR for the presence of alpha1, alpha3, beta2, beta3, and gamma

The objective of this study has been to determine which Na,K-ATPase isoforms are expressed in red blood cells and whether kinetic differences in the uncoupled sodium efflux mode between the human red blood cell Na,K-ATPase and other preparations can be explained by differences in the underlying subunit composition. To this end, human reticulocyte RNA was isolated, reverse transcribed, amplified by PCR and appropriate primers, and sequenced. Primers from highly conserved areas as well as isoform-specific primers were used. The alpha1 and alpha3 isoforms of the alpha subunit, and the beta2 and beta3 isoforms of the beta subunit were found. The complete coding regions of the cDNAs for the reticulocyte subunits were sequenced from overlapping PCR fragments. No difference was found between the reticulocyte isoforms and the ones already known. The fact that we found beta2 but not beta1 in reticulocyte single-stranded cDNA, and beta1 but not beta2 in a leukocyte library indicates that leukocyte contamination of our reticulocyte preparation was negligible. Analysis of a human bone marrow library showed that alpha1, alpha2, and alpha3 as well as all three beta isoforms were present. The extent to which the kinetic properties of uncoupled sodium efflux might depend on different isoform combinations is not yet known.     

Requirement of vascular integrin alpha v beta 3 for angiogenesis

Angiogenesis depends on the adhesive interactions of vascular cells. The adhesion receptor integrin alpha v beta 3 was identified as a marker of angiogenic vascular tissue. Integrin alpha v beta 3 was expressed on blood vessels in human wound granulation tissue but not in normal skin, and it showed a fourfold increase in expression during angiogenesis on the chick chorioallantoic membrane. In the latter assay, a monoclonal antibody to alpha v beta 3 blocked angiogenesis induced by basic fibroblast growth factor, tumor necrosis factor-alpha, and human melanoma fragments but had no effect on preexisting vessels. These findings suggest that alpha v beta 3 may be a useful therapeutic target for diseases characterized by neovascularization.     

Biochemical characterization of human collagenase-3

The cDNA of a novel matrix metalloproteinase, collagenase-3 (MMP-13) has been isolated from a breast tumor library (Freije, J. M. P., Dicz-Itza, I., Balbin, M., Sanchez, L. M., Blasco, R., Tolivia, J., and López-Otin, C. (1994) J. Biol. Chem. 269, 16766-16773), and a potential role in tumor progression has been proposed for this enzyme. In order to establish the possible role of collagenase-3 in connective tissue turnover, we have expressed and purified recombinant human procollagenase-3 and characterized the enzyme biochemically. The purified procollagenase-3 was shown to be glycosylated and displayed a M(r) of 60,000, the N-terminal sequence being LPLPSGGD, which is consistent with the cDNA-predicted sequence. The proenzyme was activated by p-aminophenylmercuric acetate or stromelysin, yielding an intermediate form of M(r) 50,000, which displayed the N-terminal sequence L58EVTGK. Further processing resulted in cleavage of the Glu84-Tyr85 peptide bond to the final active enzyme (M(r) 48,000). Trypsin activation of procollagenase-3 also generated a Tyr85 N terminus, but it was evident that the C-terminal domain was rapidly lost, and hence the collagenolytic activity diminished. Analysis of the substrate specificity of collagenase-3 revealed that soluble type II collagen was preferentially hydrolyzed, while the enzyme was 5 or 6 times less efficient at cleaving type I or III collagen. Fibrillar type I collagen was cleaved with comparable efficiency to the fibroblast and neutrophil collagenases (MMP-1 and MMP-8), respectively. Unlike these collagenases, gelatin and the peptide substrates Mea-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 and Mca-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2 were efficiently hydrolyzed as well, as would be predicted from the similarities between the active site sequence of collagenase-3 (MMP-13) and the gelatinases A and B. Active collagenase-3 was inhibited in a 1:1 stoichiometric fashion by the tissue inhibitors of metalloproteinases, TIMP-1, TIMP-2, and TIMP-3. These results suggest that in vivo collagenase-3 could play a significant role in the turnover of connective tissue matrix constituents.     

Differential effects of the integrins alpha9beta1, alphavbeta3, and alphavbeta6 on cell proliferative responses to tenascin. Roles of the beta subunit extracellular and cytoplasmic domains

Members of the integrin family manifest considerable overlap in ligand specificity, and many cells have the capacity to express multiple integrin receptors for the same ligand. For example, at least 5 different integrins recognize tenascin as a ligand, and 4 of these bind to the same region of the protein, the third fibronectin type III repeat (TNfn3). We utilized colon carcinoma cells (SW480) that do not normally attach to TNfn3 to examine the possibility that ligation of different integrin receptors for this ligand would induce different effects on cell behavior and intracellular signaling. Heterologous expression of the tenascin receptors alphavbeta3 and alpha9beta1 produced comparable effects on cell adhesion and spreading on TNfn3, but alphavbeta3-transfectants proliferated considerably better on each concentration examined. alphavbeta6-transfectants attached (although less avidly), but completely failed to spread or proliferate. Expression of a chimeric beta subunit composed of the beta3 extracellular domain fused to the beta6 transmembrane and cytoplasmic domains resulted in adhesion and spreading similar to that seen with beta3-transfectants, but considerably less proliferation. When the same cell lines were plated on fibronectin, alphavbeta6-transfectants spread and proliferated as well as cells transfected with the chimeric beta3/beta6 subunit, but, again, neither cell line proliferated as well as cells expressing alphavbeta3. Cell proliferation was always associated with spreading and with phosphorylation of the focal adhesion kinase, paxillin, and the mitogen-activated kinase, Erk2, but cell attachment in the absence of spreading or proliferation was not associated with phosphorylation of any of these proteins. These data suggest that different integrin receptors for a single ligand can produce markedly different effects on cell proliferation, and that both the extracellular and cytoplasmic domains of integrin beta subunits contribute to these differences.     

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.     

Rat neutrophils express alpha4 and beta1 integrins and bind to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1)

The alpha4 integrins, which are constitutively expressed on all human leukocyte subtypes except neutrophils, interact with vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule (MAdCAM-1) on endothelium to mediate selective recruitment of leukocyte subpopulations, other than neutrophils, to sites of inflammation. However, here we report that a different paradigm of leukocyte recruitment may exist in the rat. Flow cytometric analysis of rat neutrophils using a panel of monoclonal antibodies which recognize rat alpha4 and beta1 integrins showed consistent, low levels of expression. Although alpha4 was expressed at lower levels on neutrophils than all other rat leukocytes, this level of expression was sufficient to mediate significant levels of alpha4- and beta1-dependent neutrophil adhesion to rat and human VCAM-1, and alpha4-dependent, but beta1-independent, adhesion to human MAdCAM-1. These data suggest that rat neutrophils, unlike other species, may use alpha4 integrins to traffic to sites of inflammation in vivo.     

Stimulation of tyrosine phosphorylation after ligation of beta7 and beta1 integrins on human B cells

B lymphocytes express several members of the integrin family of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. In addition to beta1 integrins, predominantly alpha4 beta1, mature B cells also express alpha4 beta7, which is a receptor for vascular cell adhesion molecule-1 and fibronectin, and is also involved in the homing of B cells to mucosal sites through binding to a third ligand, mucosal address in cell adhesion molecule-1. Here we describe that crosslinking of alpha4 beta7 integrins on B cell lines and normal tonsillar B cells, induces tyrosine phosphorylation of multiple substrates of 105-130 kD, indicating that beta7 integrin plays a role as signaling molecule in B cells. This pattern of phosphorylated proteins was very similar to that induced following ligation of alpha4 beta1. Interestingly, ligation of alpha5 beta1 or alpha6 beta1 also stimulated the 105-125 kD group of phosphorylated proteins, whereas ligation of beta2 integrins did not. The focal adhesion tyrosine kinase p125FAK was identified as one of these substrates. Beta1 or beta7 mediated tyrosine phosphorylations were markedly decreased when the microfilament assembly was inhibited by cytochalasin B. These results suggest that intracellular signals initiated by different integrins in B cells may converge, to similar cytoskeleton-dependent tyrosine phosphorylated proteins.     

Expression of transforming growth factors beta-1, beta 2 and beta 3 in human bladder carcinomas

We previously detected elevated transforming growth factor beta-1 (TGF-beta1) serum levels in patients with invasive bladder carcinomas. In this study, we therefore investigated whether elevated serum levels correlate with enhanced TGF-beta expression in human bladder tumours. mRNA levels of TGF-beta1, -beta2 and -beta3 were reduced in bladder tumour tissue to 86%, 68% and 56%, respectively, of the levels in normal urothelium. On the other hand, TGF-beta1 protein levels were found to be higher in superficial tumours (Ta-T1) (mean level of 0.153 ng mg(-1)) and in invasive T2/T3 tumours (mean level of 0.104 ng mg(-1)) compared with normal urothelium (mean level of 0.065 ng mg(-1)). Invasive T4 tumours, however, contained only low amounts of TGF-beta1 (mean level of 0.02 ng mg(-1)). Neither in mean nor in individual patients were serum and tissue TGF-beta levels correlated with each other. Cell culture experiments on primary bladder cells revealed a 57% decrease in TGF-beta1 mRNA levels in tumour compared with normal epithelial cells. Tumour epithelial cells contained about two times higher levels of TGF-beta2 and TGF-beta3 mRNA than normal epithelial cells. Fibroblasts expressed about the same amount of TGF-beta1 or TGF-beta2 as epithelial cells. Yet, fibroblasts released only 19% and 13% of the amount secreted by tumour epithelial cells into the supernatant. TGF-beta3, on the other hand, was expressed by fibroblasts with higher levels than by epithelial cells. TGF-beta1 was the predominent isoform in bladder tissue and cells at protein as well as on mRNA levels indicating that TGFs-beta2 and -beta3 are of minor importance in bladder cancer. In summary, there is a lack of correlation between TGF-beta serum levels and TGF-beta expression in tumour tissue in bladder cancer.     

Postnatal changes of nicotinic acetylcholine receptor alpha 2, alpha 3, alpha 4, alpha 7 and beta 2 subunits genes expression in rat brain

We have previously demonstrated the development of acoustically reflective liposomes as a novel ultrasound contrast agent, that can be conjugated to antibodies for site specific acoustic enhancement of pathologically altered vascular tissue. The liposomes are echogenic due to the lipid composition, without gas entrapment, and have a size of less than one micron (Alkan-Onyuksel et al., 1996). When conjugated to anti-fibrinogen antibodies, the liposomes have the ability to attach to fibrin coated surfaces and thrombi in vitro as demonstrated by scanning electron microscopy and ultrasound imaging (Demos et al., 1997a). Anti-fibrinogen liposomes were shown to attach to fibrous atheroma and thrombi in a Yucatan miniswine model of induced atherosclerosis whereas liposomes conjugated to anti-intercellular adhesion molecule-1 (anti-ICAM-1) were demonstrated to target early stage atherosclerotic plaques (Demos et al., 1997b). The purpose of this study is to evaluate the binding characteristics of anti-fibrinogen liposomes in vitro under a variety of flow conditions in order to optimize the targeting ability of the immunoliposomes. Radiolabeled anti-fibrinogen liposomes were applied to fibrin coated filter paper and placed in a flow circuit under controlled flow conditions. Flow conditions were altered to study the effects of different shear stresses, temperature, plasma flow and pulsatile flow on the retention of liposomes to fibrin after set time periods. The retention of liposomes conjugated to polyclonal and monoclonal antibodies as well as Fab fragments made from monoclonal antibodies were compared. The binding characteristics of liposomes conjugated to different quantities of polyclonal antibodies were analyzed. At physiological shear stress of 1.5 N/m2 (15 dynes/cm2) over 70% of the liposomes remained attached to fibrin after two hours. A smaller and greater portion of the liposomes remained attached at higher and lower shear stresses respectively. Plasma components and temperature had no effect on liposomal retention whereas pulsatile flow resulted in a slight reduction in binding. Monoclonal antibodies showed a slight trend of reduced retention to fibrin over time as compared with polyclonal antibodies and Fab fragments. The quantity of antibody conjugated to the liposomes plays a role in liposome retention as demonstrated by the reduction in liposome retention caused by reducing the quantity of antibody conjugated to the liposomes. Anti-fibrinogen liposomes were retained to the fibrin surface to a large extent under all flow conditions likely to occur in vivo and therefore can provide site specific ultrasound contrast for a long enough time period to allow for imaging after injection.     

Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics

The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.