Chapter Nine - Cellular Roles of Beta-Arrestins as Substrates and Adaptors of Ubiquitination and Deubiquitination.

ß-Arrestin1 and ß-arrestin2 are homologous adaptor proteins that are ubiquitously expressed in mammalian cells. They belong to a four-member family of arrestins that regulate the vast family of seven-transmembrane receptors that couple to heterotrimeric G proteins (7TMRs or GPCRs), and that modulate 7TMR signal transduction. ß-Arrestins were originally identified in the context of signal inhibition via the 7TMRs because they competed with and thereby blocked G protein coupling to 7TMRs. Currently, in addition to their role as desensitizers of signaling, ß-arrestins are appreciated as multifunctional adaptors that mediate trafficking and signal transduction of not only 7TMRs, but a growing list of additional receptors, ion channels, and nonreceptor proteins. ß-Arrestins' interactions with their multifarious partners are based on their dynamic conformational states rather than particular domain-domain interactions. ß-Arrestins adopt activated conformations upon 7TMR association. In addition, ß-arrestins undergo various posttranslational modifications that are choreographed by activated 7TMRs, including phosphorylation, ubiquitination, acetylation, nitrosylation, and SUMOylation. Ubiquitination of ß-arrestins is critical for their high-affinity interaction with 7TMRs as well as with endocytic adaptor proteins and signaling kinases. ß-Arrestins also function as critical adaptors for ubiquitination and deubiquitination of various cellular proteins, and thereby affect the longevity of signal transducers and the intensity of signal transmission.

Polymorphisms of the tumor suppressor gene LSAMP are associated with left main coronary artery disease.

Previous association mapping on chromosome 3q13-21 detected evidence for association at the limbic system-associated membrane protein (LSAMP) gene in individuals with late-onset coronary artery disease (CAD). LSAMP has never been implicated in the pathogenesis of CAD. We sought to thoroughly characterize the association and the gene. Non-redundant single nucleotide polymorphisms (SNPs) across the gene were examined in an initial dataset (168 cases with late-onset CAD, 149 controls). Stratification analysis on left main CAD (N = 102) revealed stronger association, which was further validated in a validation dataset (141 cases with left main CAD, 215 controls), a third control dataset (N = 255), and a family-based dataset (N = 2954). A haplotype residing in a novel alternative transcript of the LSAMP gene was significant in all independent case-control datasets (p = 0.0001 to 0.0205) and highly significant in the joint analysis (p = 0.00004). Lower expression of the novel alternative transcript was associated with the risk haplotype (p = 0.0002) and atherosclerosis burden in human aortas (p = 0.0001). Furthermore, silencing LSAMP expression in human aortic smooth muscle cells (SMCs) substantially augmented SMC proliferation (p<0.01). Therefore, the risk conferred by the LSAMP haplotype appears to be mediated by LSAMP down-regulation, which may promote SMC proliferation in the arterial wall and progression of atherosclerosis.

Serine 232 of the alpha(2A)-adrenergic receptor is a protein kinase C-sensitive effector coupling switch.

alpha(2)-adrenergic receptors (alpha(2)AR) couple to multiple effectors including adenylyl cyclase and phospholipase C. We hypothesized that signaling selectivity to these effectors is dynamically directed by kinase-sensitive domains within the third intracellular loop of the receptor. Substitution of Ala for Ser232, which is in the N-terminal region of this loop in the alpha(2A)AR, resulted in a receptor that was markedly uncoupled ( approximately 82% impairment) from stimulation of inositol phosphate accumulation while the capacity to inhibit adenylyl cyclase remained relatively intact. In S232A alpha(2A)AR transfected cell membranes, agonist-promoted [(35)S]GTPgammaS binding was reduced by approximately 50%. Coexpression of modified G proteins rendered insensitive to pertussis toxin revealed that the S232A receptor was uncoupled from both G(i) and G(o). S232 is a potential PKC phosphorylation site, and whole cell phosphorylation studies showed that the mutant had depressed phosphorylation compared to wild type (1.3- vs 2.1-fold/basal). Consistent with S232 directing coupling to phospholipase C, PMA exposure resulted in approximately 67% desensitization of agonist-promoted inositol phosphate accumulation without significantly affecting inhibition of adenylyl cyclase. The dominant effect of mutation or phosphorylation at this site on inositol phosphate as compared to cAMP signaling was found to most likely be due to the low efficiency of signal transduction via phospholipase C vs adenylyl cyclase. Taken together, these results indicate that S232 acts as a selective, PKC-sensitive, modulator of effector coupling of the alpha(2A)AR to inositol phosphate stimulation. This represents one mechanism by which cells route stimuli directed to multifunctional receptors to selected effectors so as to attain finely targeted signaling.

Overexpression of G protein-coupled receptor kinase-2 in smooth muscle cells attenuates mitogenic signaling via G protein-coupled and platelet-derived growth factor receptors.

BACKGROUND: Neointimal hyperplasia involves activation of smooth muscle cells (SMCs) by several G protein-coupled receptor (GPCR) agonists, including endothelin-1, angiotensin II, thrombin, and thromboxane A(2). Signaling of many GPCRs is diminished by GPCR kinase-2 (GRK2). We therefore tested whether overexpression of GRK2 in SMCs could diminish mitogenic signaling elicited by agonists implicated in the pathogenesis of neointimal hyperplasia. METHODS AND RESULTS: Overexpression of GRK2 was achieved in primary rabbit aortic SMCs with a recombinant adenovirus. Control SMCs were infected with an empty vector adenovirus. Inositol phosphate responses to endothelin-1, angiotensin II, thrombin agonist peptide, and platelet-derived growth factor (PDGF) were attenuated by 37% to 72% in GRK2-overexpressing cells (P<0.01), but the response to the thromboxane A(2) analogue U46619 was unaffected. GRK2 also inhibited SMC [(3)H]thymidine incorporation stimulated not only by these agonists (by 30% to 60%, P<0.01) but also by 10% FBS (by 35%, P<0. 05). However, GRK2 overexpression had no effect on epidermal growth factor-induced [(3)H]thymidine incorporation. Agonist-induced tyrosine phosphorylation of the PDGF-beta receptor, but not the epidermal growth factor receptor, was reduced in GRK2-overexpressing SMCs. GRK2 overexpression also reduced SMC proliferation in response to endothelin-1, PDGF, and 10% FBS by 62%, 51%, and 29%, respectively (P<0.01), without any effect on SMC apoptosis. CONCLUSIONS: GRK2 overexpression diminishes SMC mitogenic signaling and proliferation stimulated by PDGF or agonists for several GPCRs. Gene transfer of GRK2 may therefore be therapeutically useful for neointimal hyperplasia.

G protein-coupled receptor kinases.

G protein-coupled receptor kinases (GRKs) constitute a family of six mammalian serine/threonine protein kinases that phosphorylate agonist-bound, or activated, G protein-coupled receptors (GPCRs) as their primary substrates. GRK-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling, or desensitization. This review focuses on the regulation of GRK activity by a variety of allosteric and other factors: agonist-stimulated GPCRs, beta gamma subunits of heterotrimeric GTP-binding proteins, phospholipid cofactors, the calcium-binding proteins calmodulin and recoverin, posttranslational isoprenylation and palmitoylation, autophosphorylation, and protein kinase C-mediated GRK phosphorylation. Studies employing recombinant, purified proteins, cell culture, and transgenic animal models attest to the general importance of GRKs in regulating a vast array of GPCRs both in vitro and in vivo.

Phosphorylation and desensitization of human endothelin A and B receptors. Evidence for G protein-coupled receptor kinase specificity.

Although endothelin-1 can elicit prolonged physiologic responses, accumulating evidence suggests that rapid desensitization affects the primary G protein-coupled receptors mediating these responses, the endothelin A and B receptors (ETA-R and ETB-R). The mechanisms by which this desensitization proceeds remain obscure, however. Because some intracellular domain sequences of the ETA-R and ETB-R differ substantially, we tested the possibility that these receptor subtypes might be differentially regulated by G protein-coupled receptor kinases (GRKs). Homologous, or receptor-specific, desensitization occurred within 4 min both in the ETA-R-expressing A10 cells and in 293 cells transfected with either the human ETA-R or ETB-R. In 293 cells, this desensitization corresponded temporally with agonist-induced phosphorylation of each receptor, assessed by receptor immunoprecipitation from 32Pi-labeled cells. Agonist-induced receptor phosphorylation was not substantially affected by PKC inhibition but was reduced 40% (p << 0.03) by GRK inhibition, effected by a dominant negative GRK2 mutant. Inhibition of agonist-induced phosphorylation abrogated agonist-induced ETA-R desensitization. Overexpression of GRK2, -5, or -6 in 293 cells augmented agonist-induced ET-R phosphorylation approximately 2-fold (p << 0.02), but each kinase reduced receptor-promoted phosphoinositide hydrolysis differently. While GRK5 inhibited ET-R signaling by only approximately 25%, GRK2 inhibited ET-R signaling by 80% (p << 0.01). Congruent with its superior efficacy in suppressing ET-R signaling, GRK2, but not GRK5, co-immunoprecipitated with the ET-Rs in an agonist-dependent manner. We conclude that both the ETA-R and ETB-R can be regulated indistinguishably by GRK-initiated desensitization. We propose that because of its affinity for ET-Rs demonstrated by co-immunoprecipitation, GRK2 is the most likely of the GRKs to initiate ET-R desensitization.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

Phosphorylation of the type 1A angiotensin II receptor by G protein-coupled receptor kinases and protein kinase C.

The type 1A angiotensin II receptor (AT1A-R), which mediates cardiovascular effects of angiotensin II, has been shown to undergo rapid agonist-induced desensitization. We investigated the potential role of second messenger-activated kinases and G protein-coupled receptor kinases (GRKs) in the regulation of this receptor. In 293 cells transfected with the AT1A-R, a 3-min challenge with angiotensin II engendered a 46% decrease in subsequent angiotensin II-stimulated phosphoinositide hydrolysis in intact cells. This agonist-induced desensitization correlated temporally and dose-dependently with the phosphorylation of the receptor to a stoichiometry of 1 mol of phosphate/mol of receptor, as assessed by immunoprecipitation of receptors from cells metabolically labeled with 32Pi. Agonist-induced receptor phosphorylation was reduced by 40-50% by either overexpression of a dominant negative K220R mutant GRK2 or treatment of the cells with the protein kinase C (PKC) inhibitor staurosporine, in a virtually additive fashion. Cellular overexpression of GRK2K220R not only inhibited agonist-induced AT1A-R phosphorylation, but also prevented receptor desensitization, as assessed by angiotensin II-stimulated GTPase activity in membranes prepared from agonist-treated and control cells. In contrast, PKC inhibition by staurosporine did not affect homologous desensitization of the AT1A-R. Overexpression of GRKs 2, 3, or 5 significantly augmented the agonist-induced AT1A-R phosphorylation 1.5- to 1.7-fold (p < 0.001). These findings suggest a role for receptor phosphorylation by one or several GRKs in the rapid agonist-induced desensitization of the AT1A-R.

Desensitization of G protein-coupled receptors.

Waning responsiveness to continuous or repeated stimulation constitutes the phenomenon of desensitization, which pervades biological systems. Over the last several years, molecular mechanisms for desensitization of cellular signaling through G protein-coupled receptors have been delineated, particularly at the level of the receptors themselves. This review focuses on those aspects of G protein-coupled receptor desensitization which occur within minutes of agonist exposure. Agonist-dependent desensitization of these receptors can reduce their signaling responsiveness to maximum stimulation by up to 70-80%; indeed, in some receptor systems, the process of receptor desensitization appears to effect the termination of the cellular signaling response. Agonist-induced desensitization involves phosphorylation of G protein-coupled receptors by two currently recognized classes of serine/threonine protein kinases. Second messenger-dependent kinases, phosphorylating a variety of proteins, mediate a generalized cellular hyporesponsiveness termed heterologous desensitization. G protein-coupled receptor kinases (GRKs) phosphorylate specifically agonist-occupied, or activated, receptors, and thereby initiate receptor-specific, or homologous, desensitization. GRK-mediated receptor phosphorylation facilitates the binding of an inhibitory arrestin protein to the phosphorylated receptor, an event which substantially impairs receptor signaling. The GRK family comprises six, and the arrestin family comprises four known members. Each GRK demonstrates the ability to phosphorylate a limited number of model receptor substrates, but the widespread expression of several GRKs and the two somatic arrestins suggest that GRK-initiated homologous desensitization is of general importance to a wide range of G protein-coupled receptors. Exploration of the substrate specificity for GRKs and the ligand specificity for arrestins remains in its early stages. Currently, GRKs can most readily be differentiated by the mechanisms each employs to associate with the plasma membrane. Accumulating data from human disease states and transgenic mice attest to the physiologic significance of GRK-initiated receptor desensitization.

Agonist-dependent phosphorylation of the mouse delta-opioid receptor: involvement of G protein-coupled receptor kinases but not protein kinase C.

With chronic opiate use, opioid receptor desensitization may be one of the important mechanisms underlying the development of opiate tolerance and addiction. Opioid receptors belong to the G protein-coupled receptor superfamily. In this study, the mouse delta-opioid receptor (delta OR) was used in a model system to investigate the role of opioid receptor phosphorylation in receptor desensitization. When expressed in 293 cells and exposed to agonist, the delta OR underwent receptor-specific desensitization within 10 min. This agonist-induced desensitization corresponded temporally to a 3-fold increase in receptor phosphorylation. Phorbol ester, but not forskolin, also stimulated phosphorylation of the delta OR in 293 cells. Although down-regulation of protein kinase C failed to affect agonist-induced receptor phosphorylation, it abolished phorbol ester-induced receptor phosphorylation. Agonist-induced delta OR phosphorylation must therefore involve kinases other than protein kinase C. Whereas overexpression of a dominant negative mutant (K220R) of beta-adrenergic receptor kinase-1 (beta ARK1) in 293 cells significantly reduced agonist-dependent phosphorylation of the delta OR, overexpression of beta ARK1 or G protein-coupled receptor kinase-5 significantly enhanced this phosphorylation. Concordantly, beta ARK1-K220R overexpression reduced agonist-dependent delta OR desensitization, whereas beta ARK1 overexpression enhanced this densensitization. We conclude that short term desensitization of the delta OR involves phosphorylation of the receptor by one or more G protein-coupled receptor kinases.

Phosphorylation and desensitization of the human beta 1-adrenergic receptor. Involvement of G protein-coupled receptor kinases and cAMP-dependent protein kinase.

Persistent stimulation of the beta 1-adrenergic receptor (beta 1AR) engenders, within minutes, diminished responsiveness of the beta 1 AR/adenylyl cyclase signal transduction system. This desensitization remains incompletely defined mechanistically, however. We therefore tested the hypothesis that agonist-induced desensitization of the beta 1AR (like that of the related beta 2AR) involves phosphorylation of the receptor itself, by cAMP-dependent protein kinase (PKA) and the beta-adrenergic receptor kinase (beta ARK1) or other G protein-coupled receptor kinases (GRKs). Both Chinese hamster fibroblast and 293 cells demonstrate receptor-specific desensitization of the beta 1 AR within 3-5 min. Both cell types also express beta ARK1 and the associated inhibitory proteins beta-arrestin-1 and beta-arrestin-2, as assessed by immunoblotting. Agonist-induced beta 1AR desensitization in 293 cells correlates with a 2 +/- 0.3-fold increase in phosphorylation of the beta 1AR, determined by immunoprecipitation of the beta 1AR from cells metabolically labeled with 32P(i). This agonist-induced beta 1AR phosphorylation derives approximately equally from PKA and GRK activity, as judged by intact cell studies with kinase inhibitors or dominant negative beta ARK1 (K220R) mutant overexpression. Desensitization, likewise, is reduced by only approximately 50% when PKA is inhibited in the intact cells. Overexpression of rhodopsin kinase, beta ARK1, beta ARK2, or GRK5 significantly increases agonist-induced beta 1AR phosphorylation and concomitantly decreases agonist-stimulated cellular cAMP production (p < 0.05). Furthermore, purified beta ARK1, beta ARK2, and GRK5 all demonstrate agonist-dependent phosphorylation of the beta 1AR. Consistent with a GRK mechanism, receptor-specific desensitization of the beta 1AR was enhanced by overexpression of beta-arrestin-1 and -2 in transfected 293 cells. We conclude that rapid agonist-induced desensitization of the beta 1AR involves phosphorylation of the receptor by both PKA and at least beta ARK1 in intact cells. Like the beta 2AR, the beta 1AR appears to bind either beta-arrestin-1 or beta-arrestin-2 and to react with rhodopsin kinase, beta ARK1, beta ARK2, and GRK5.

A highly conserved tyrosine residue in G protein-coupled receptors is required for agonist-mediated beta 2-adrenergic receptor sequestration.

An aromatic residue, tyrosine 326 in the prototypical human beta 2-adrenergic receptor, exists in a highly conserved sequence motif in virtually all members of the G protein-coupled receptor family. The potential role of this conserved aromatic amino acid residue in the cellular processes of sequestration (a rapid internalization of the surface receptor) and down-regulation (a slower loss of total cellular receptors) associated with agonist-mediated desensitization of the beta 2-adrenergic receptor was assessed by replacing tyrosine residue 326 with an alanine residue (beta 2AR-Y326A). This mutation completely abolishes agonist-mediated receptor sequestration without affecting the ability of the receptor to activate maximally adenylyl cyclase, to undergo rapid desensitization, and to down-regulate in response to agonist. The only other major change associated with the mutated receptor is a complete loss of the ability to resensitize following rapid desensitization. These results imply that this tyrosine residue, which is part of a highly conserved sequence motif in G protein-coupled receptors, may be responsible for their agonist-mediated sequestration and that sequestration and down-regulation of the receptor are dissociable phenomena. The lack of resensitization in the sequestration-defective beta 2-adrenergic receptor mutant strongly suggests that the sequestration pathway is an important mechanism by which cells re-establish the normal responsiveness of G protein-coupled receptors following the removal of agonist.

Inhibition of thrombin receptor signaling by a G-protein coupled receptor kinase. Functional specificity among G-protein coupled receptor kinases.

The thrombin receptor, a member of the seven membrane-spanning superfamily of G-protein coupled receptors, is activated by an irreversible proteolytic mechanism, but signaling by activated thrombin receptors shuts off soon after receptor activation. This shut-off mechanism is thought to be required for concentration-dependent responses to thrombin and an important determinant of the cell's sensitivity to thrombin. We report that the thrombin receptor is rapidly phosphorylated upon activation, consistent with the action of a G-protein-coupled receptor kinase. Moreover, the G-protein coupled receptor kinase BARK2 (beta-adrenergic receptor kinase 2) blocked signaling by thrombin receptors coexpressed in Xenopus oocytes. In this system, rhodopsin kinase was inactive and BARK1 was markedly less effective than BARK2. Thrombin receptor mutants which lacked potential serine and threonine phosphorylation sites in the receptor's cytoplasmic tail were insensitive to inhibition by exogenous BARK2 but did confer concentration-dependent responses to thrombin. Our studies demonstrate that a G-protein coupled receptor kinase can shut off thrombin receptor signaling but that additional mechanism(s) for terminating signaling exist. These studies also reveal functional specificity among G-protein coupled receptor kinases in a novel in vivo reconstitution system and show that heterologous expression of these kinases can be used to manipulate cellular responsiveness.

Effects of ocular carteolol and timolol on plasma high-density lipoprotein cholesterol level.

Fifty-eight healthy, normolipidemic adult men participated in a prospective, masked, randomized crossover study designed to compare the effects of two topical nonselective beta-adrenergic antagonists, carteolol and timolol, on plasma high-density lipoprotein cholesterol levels. Two eight-week treatment periods were separated by an eight-week drug-free period. Carteolol 1.0% or timolol 0.5% was used, one drop twice daily, in both eyes without nasolacrimal occlusion. Fresh plasma was assayed for levels of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and apolipoproteins A-I and B-100. With indistinguishable effects on intraocular pressure, carteolol and timolol induced different (P = .013) decrements in high-density lipoprotein cholesterol levels. Carteolol treatment decreased high-density lipoprotein cholesterol levels by 3.3% (-0.04 mmol/l) and raised the ratio of total to high-density lipoprotein cholesterol levels by 4.0% (0.15 unit); timolol treatment decreased high-density lipoprotein cholesterol levels by 8.0% (-0.10 mmol/l) and raised the ratio of total to high-density lipoprotein cholesterol levels by 10.0% (0.37 unit). There was no differential drug effect on the other lipid variables measured. Ocular nonselective beta-adrenergic antagonist therapy can produce clinically relevant decrements in high-density lipoprotein cholesterol levels in healthy men.

Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.

Secreted extracellular domains of macrophage scavenger receptors form elongated trimers which specifically bind crocidolite asbestos.

Macrophage scavenger receptors, which have been implicated in the development of atherosclerosis and other macrophage-mediated events, are trimeric integral membrane glycoproteins whose extracellular domains have been predicted to include alpha-helical coiled-coil, collagenous and globular structures. To elucidate further the structural and functional properties of these receptors, we generated transfected Chinese hamster ovary cells which express secreted extracellular domains of the type I and type II bovine scavenger receptors and developed a solid-phase bead-binding assay to assess their ligand-binding properties. The secreted receptors exhibited the distinctive high-affinity, broad polyanionic ligand-binding specificity and the pH dependence of binding which characterize the membrane-anchored cell-surface forms of the receptors. Both the type I and type II secreted receptors were trimeric glycoproteins comprising disulfide-linked dimers and noncovalently associated monomers. Gel filtration and glycerol-gradient centrifugation established that the type II trimers were highly elongated and did not associate into higher order oligomers at the low concentrations used in these experiments. Crocilodite asbestos, which is phagocytosed by alveolar macrophages and can cause asbestosis and mesothelioma, bound efficiently to secreted type I receptors and less well to the type II receptors. This binding was specific in that it was competed by a variety of well established scavenger receptor ligands but not by negative controls. These studies have identified a new type of insoluble scavenger receptor ligand, and have raised the possibility that scavenger receptors may play a role in mediating the physiological and pathological interactions of inspired particles with alveolar macrophages.

The type I and type II bovine scavenger receptors expressed in Chinese hamster ovary cells are trimeric proteins with collagenous triple helical domains comprising noncovalently associated monomers and Cys83-disulfide-linked dimers.

Scavenger receptors have been implicated in the development of atherosclerosis and other macrophage-associated functions. The structures and processing of type I and type II bovine macrophage scavenger receptors were examined using polyclonal anti-receptor antibodies. Pulse/chase metabolic labeling experiments showed that both types of scavenger receptors expressed in Chinese hamster ovary (CHO) cells behaved as typical cell surface membrane glycoproteins. They were synthesized as endoglycosidase H-sensitive precursors which were converted to endoglycosidase H-resistant mature forms expressed on the cell surface. The reduced precursor and mature forms were doublets on sodium dodecyl sulfate-gel electrophoresis, primarily because of heterogeneous N-glycosylation. The approximate molecular sizes were: type I precursor, 65/63 kDa; type I mature, 82/76 kDa; type II precursor, 57/53 kDa; and type II mature, 72/65 kDa. During post-translational processing, the cysteine-rich C terminus (SRCR domain) of some of the type I receptors was proteolytically removed to form a relatively stable, approximately 69-kDa degradation product. Type II receptors differ from type I receptors in that they do not have SRCR domains and an analogous proteolytic cleavage was not observed. Several experiments provided strong evidence that the Gly-X-Y-repeat domains in the scavenger receptors oligomerize into collagenous triple helices. For example, alpha,alpha'-dipyridyl, an inhibitor of the collagen-modifying enzymes prolyl and lysyl hydroxylases, interfered with both the kinetics and nature of post-translational receptor processing, and both precursor and mature forms of the receptors in intact cells could be cross-linked with difluorodinitrobenzene into reduction-resistant trimers. In intact cells, precursor receptor trimers (type I, 198 kDa; type II, 176 kDa) were assembled in the endoplasmic reticulum by the noncovalent association of monomers and Cys83-disulfide-linked dimers (type I, 129 kDa; type II, 119 kDa). When cells were lysed in the absence of the sulfhydryl trapping agent iodoacetamide, oxidation of the side chain of Cys17 in the cytoplasmic domain leads to the artifactual formation of reduction-sensitive covalently linked trimers. The approximate masses of the mature dimer and trimer forms were 162 and 237 kDa for type I receptors and 147 and 219 kDa for type II receptors. Cys83-disulfide-linked dimer formation was not required for function because mutant receptors (Cys83----Gly83) assembled into trimers of noncovalently associated monomers and exhibited normal receptor activity. Treatment of cells with difluorodinitrobenzene cross-linked some of the receptors into complexes larger than trimers, raising the possibility that the trimers may assemble into higher order oligomers.

Expression of type I and type II bovine scavenger receptors in Chinese hamster ovary cells: lipid droplet accumulation and nonreciprocal cross competition by acetylated and oxidized low density lipoprotein.

Type I and type II scavenger receptors, which have been implicated in the development of atherosclerosis and other macrophage-associated functions, differ only by the presence in the type I receptor of an extracellular cysteine-rich C-terminal domain. Stable Chinese hamster ovary (CHO) cell transfectants expressing high levels of either the type I or type II bovine scavenger receptors have been generated. Type I and type II receptors in these cells mediated high-affinity saturable endocytosis of both 125I-labeled acetylated low density lipoprotein (LDL) and 125I-labeled oxidized LDL with the distinctive broad ligand specificity characteristic of scavenger receptors. After incubation for 2 days with acetylated LDL, the transfected cells accumulated oil red O-staining lipid droplets reminiscent of those in macrophage foam cells, whereas untransfected CHO cells did not. Thus, macrophage-specific gene products other than the scavenger receptor are not required for modified-LDL-induced intracellular lipid accumulation. In transfected cells, acetylated LDL efficiently competed for both its own endocytosis and that of oxidized LDL. In contrast, oxidized LDL competed effectively for its own endocytosis but only poorly for that of acetylated LDL. This nonreciprocal cross competition suggests that these ligands may bind to nonidentical but interacting sites on a single receptor. Results were similar for transfectants expressing either type I or type II scavenger receptors. Therefore, the nonreciprocal cross competition previously reported for cultured peritoneal macrophages may not be the result of differences between the type I and type II receptors. The nonreciprocal cross competition seen in the transfected CHO cells differs from that previously observed with cultured macrophages.