D1 and D2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons.

The striatum, which is the major component of the basal ganglia in the brain, is regulated in part by dopaminergic input from the substantia nigra. Severe movement disorders result from the loss of striatal dopamine in patients with Parkinson's disease. Rats with lesions of the nigrostriatal dopamine pathway caused by 6-hydroxydopamine (6-OHDA) serve as a model for Parkinson's disease and show alterations in gene expression in the two major output systems of the striatum to the globus pallidus and substantia nigra. Striatopallidal neurons show a 6-OHDA-induced elevation in their specific expression of messenger RNAs (mRNAs) encoding the D2 dopamine receptor and enkephalin, which is reversed by subsequent continuous treatment with the D2 agonist quinpirole. Conversely, striatonigral neurons show a 6-OHDA-induced reduction in their specific expression of mRNAs encoding the D1 dopamine receptor and substance P, which is reversed by subsequent daily injections of the D1 agonist SKF-38393. This treatment also increases dynorphin mRNA in striatonigral neurons. Thus, the differential effects of dopamine on striatonigral and striatopallidal neurons are mediated by their specific expression of D1 and D2 dopamine receptor subtypes, respectively.

Oligonucleotides antisense to the interleukin 1 receptor mRNA block the effects of interleukin 1 in cultured murine and human fibroblasts and in mice.

Phosphodiester and phosphorothioate oligodeoxynucleotides (18 mers) were constructed antisense to sequences of the recently cloned murine and human IL-1 receptors. Murine antisense oligonucleotides inhibited IL-1-stimulated PGE2 synthesis by murine fibroblasts in culture in a time (days) and concentration-dependent (3 microM-30 microM) fashion. Murine sense oligonucleotide and an oligonucleotide antisense to human IL-1 receptor were without effect. Moreover, murine antisense oligonucleotides did not affect tumor necrosis factor- or bradykinin-stimulated PGE2 synthesis by murine fibroblasts. Similarly, antisense oligonucleotides to the human, but not the murine, IL-1 receptor inhibited IL-1-stimulated PGE2 synthesis by cultured human fibroblasts. The attenuation of the cellular response to IL-1 caused by the antisense oligonucleotides correlated with a loss in cell surface receptors for IL-1, without any change in the number of bradykinin receptors on these cells. When antisense oligonucleotides were encapsulated in liposomes, they blocked completely the appearance of newly synthesized IL-1 receptors and IL-1-stimulated PGE2 synthesis. In mice, subcutaneous injection with an oligonucleotide antisense to the murine IL-1 receptor markedly inhibited the infiltration of neutrophils in response to subsequent injection of IL-1. These data suggest that antisense oligodeoxynucleotides may share a role in the design of antiinflammatory therapeutics.

Some properties of alkali-extracted red cell ghost membranes.

The properties of integral membrane proteins obtained by dilute alkali extraction of red cell ghosts were examined. A variety of conditions promoted the disulfide-mediated aggregation of integral membrane proteins, principally band 3. Procedural modifications which minimized aggregation were the use of EDTA and S-alkylation. Integral membrane proteins were solubilized under non-denaturing conditions using Brij 36T, a lauryl polyoxyethylene ether with an NMR-determined average chain length of 8.2 (oxyethylene) units. Detergent gel filtration revealed a chromatographic shoulder due to aggregated band 3 when membrane proteins were not alkylated. Analyses of the column profile also revealed a discrete peak for sialoglycoproteins and two phosphate peaks, an early one due to phospholipid and a later one not identified, but probably due to phosphoinositide.

Selective down-regulation of adrenergic receptor subtypes in tissues from rats with pheochromocytoma.

We have used an animal model of pheochromocytoma and radioligand-binding techniques to examine the effects of high levels of circulating norepinephrine and dopamine on adrenergic receptor subtypes in several peripheral tissues. New England Deaconess Hospital rats with transplanted pheochromocytomas were hypertensive and had levels of plasma norepinephrine and dopamine 50-fold greater than those of controls. The number of beta-adrenergic receptors in membranes prepared from the renal cortex and the left ventricle from these rats was decreased about 50%, but the animals had no significant decrease in the overall number of beta-adrenergic receptors in pulmonary membranes. beta-Receptor affinity was unaltered in animals with pheochromocytoma. Competition for [125I]iodocyanopindolol binding to beta-receptors by subtype-selective agents indicated a selective decrease of about 80% in the number of beta 1-adrenergic receptors in renal cortical and pulmonary membranes, without a decrease in beta 2-adrenergic receptor number. Rats with pheochromocytoma also had about a 70% decrease in the number of alpha 1-adrenergic receptors in membranes from renal cortex and lung, but no significant decrease in the number of alpha 1-adrenergic receptors in hepatic membranes and no decrease in the number of alpha 2-adrenergic receptors in renal cortical and hepatic membranes. These results indicate that rats in which pheochromocytomas are transplanted show adrenergic receptor subtype- and tissue-specific down-regulation. Although the selective down-regulation of alpha 1- and beta 1-adrenergic receptors may reflect a response to the preponderance of norepinephrine in these animals, the results indicate that different tissues and different adrenergic receptor subtypes may have varying susceptibility to down-regulation in response to increased circulating catecholamines in vivo.

Pharmacological characterization and region-specific expression in brain of the beta 2- and beta 3-subunits of the rat GABAA receptor.

The cDNA for a third beta-subunit of the rat GABAA receptor has been cloned using another beta-subunit, which we had previously cloned [(1989) FEBS Lett. 246, 145-148], as a probe. The approximately 8-kb cDNA for this beta-subunit (termed beta 2) encodes a protein of 474 amino acid residues that shares approximately 80% sequence identity with the rat and bovine beta 1- and beta 3-subunits. Coexpression of the cloned beta-subunit cDNA with the alpha 1-subunit cDNA of the rat GABAA receptor in Xenopus oocytes produced a functional receptor and Cl- channel with pharmacological characteristics of a GABAA receptor. In contrast to interchanging alpha-subunits [(1988) Nature 335, 76-79], exchange of beta 2- or beta 3-subunits in an alpha 1/beta receptor complex did not markedly alter the pharmacological properties of expressed receptors. In situ hybridization histochemistry with synthetic subunit-specific oligo-deoxynucleotide probes revealed a region-specific expression of alpha 1-, beta 2- and beta 3-subunit mRNAs in the rat central nervous system. These observations provide an additional molecular basis for the functional heterogeneity in the GABAA receptor complex.

Multiple GABAA receptor alpha subunit mRNAs revealed by developmental and regional expression in rat, chicken and human brain.

GABAA receptor alpha subunit transcripts were detected by Northern analysis of rat, chicken and human brain mRNA using a series of 32P-labelled antisense RNA probes derived from human alpha 1 subunit cDNAs. These alpha subunit mRNAs differ in their distribution among various brain regions in the rat and at least one species is detected primarily in fetal brain. GABAA receptor alpha 1 subunit probes encoding the putative extracellular domain detect at least five alpha subunit transcripts in rat brain, whereas probes encoding the putative intracellular domain detect only two mRNAs. These data suggest the presence in brain of multiple GABAA receptor alpha subunits having homologous extracellular domains and whose expression is regionally and developmentally regulated. These alpha subunit transcripts may encode proteins that comprise GABAA isoreceptors differing in their pharmacological and physiological properties.

Cloning and expression of a novel rat GABAA receptor.

Two full-length cDNA clones encoding alpha- and beta-subunits of a GABAA receptor have been isolated from a rat cerebral cortex cDNA library. The mature alpha-subunit protein consists of 428 amino acids with a calculated Mr of 48,680. This protein is highly homologous (approximately 99% amino acid identity) with the bovine brain alpha 1-subunit receptor [(1988) Nature 335, 76-79]. The mature rat beta-subunit receptor is a 448 amino acid polypeptide and shares approximately 80% amino acid identity with the previously characterized bovine GABAA receptor beta-subunit [(1987) Nature 328, 221-227]. Co-expression of the cloned DNA in Xenopus oocytes produces a functional receptor and ion channel with pharmacological characteristics of a GABAA receptor. GABAA alpha- and beta-subunit mRNA is detectable in the cortex, cerebellum and hippocampus.

Co-existent expression of GABAA receptor beta 2, beta 3 and gamma 2 subunit messenger RNAs during embryogenesis and early postnatal development of the rat central nervous system.

The expression of beta 1, beta 2, beta 3, gamma 2 and delta subunit messenger RNAs of the GABAA receptor was followed by in situ hybridization histochemistry using radiolabeled oligodeoxynucleotide probes in sections of embryonic (E12-21) and early postnatal (P1-5) rat. beta 2, beta 3 and gamma 2 subunit messenger RNAs were first detectable at E15 in the spinal cord (ventral > dorsal) and lower central nervous system regions (e.g. pons, medulla and thalamus). beta 3 subunit messenger RNA was abundantly expressed in olfactory bulb neurons at E15. At E17, the expression pattern of these subunit messenger RNAs continued in the lower central nervous system. In the upper central nervous system, beta 2, beta 3, and gamma 2 subunit messenger RNAs were first detectable in the outer layer of the hippocampal and entire cortical neuroepithelium. The expression for both beta 3 and gamma 2 subunit messenger RNAs increased significantly over that observed at E15, whereas beta 2 subunit messenger RNA increased to a lesser extent and was more discretely expressed in inferior colliculus, cerebellar neuroepithelium and spinal cord (ventral = dorsal). By E19, messenger RNAs for beta 2, beta 3 and gamma 2 subunits a widespread and abundant co-existent distribution throughout the central nervous system. Exceptions to this co-expression were the absence of beta 2 messenger RNA in the dentate gyrus and beta 3 messenger RNA in entorhinal cortex, areas in which they are present in adult. There was also a differential distribution of subunit messenger RNAs in developing olfactory bulb at E19-20: the glomerular cells preferentially expressed beta 3 and gamma 2 subunit messenger RNAs; the mitral cells preferentially expressed beta 2 subunit messenger RNA; inner granule cells expressed moderate levels of beta 2, beta 3 and gamma 2 subunit messenger RNAs. Expression of beta 2, beta 3 and gamma 2 messenger RNAs was also anatomically co-existent at P5. In addition, significant expression of beta 1 and delta subunit messenger RNAs was apparent in hippocampus and entorhinal cortex. The identity of the gamma 2 expressed between E15 and E21 was shown to be mostly the short isoform of gamma 2 subunit messenger RNA. Expression of both forms was evident beginning around P3-5. These results indicate that during the late embryonic and early postnatal period of development, beta 2, beta 3 and gamma 2 subunit messenger RNAs are abundantly expressed and co-localized to most central nervous system regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Method for identifying ligands activating either excitatory or inhibitory G-protein-coupled receptors by functional coexpression in Xenopus oocytes.

Xenopus oocytes devoid of their follicular enclosure provide a frequently used expression system for investigating receptors that transduce through activation of adenylyl cyclase following injection of the appropriate mRNA. However, due to a low basal activity of the cyclase they cannot be utilized to investigate receptor-mediated reductions in endogenous cAMP levels. In order to overcome this limitation, a model was designed in which test clones for such inhibitory receptors were co-expressed with a beta 2-adrenoceptor, which elevated cAMP upon exposure to isoproterenol. Following injection of mRNA to express the alpha 2 test receptor in the oocytes, marked reduction in cAMP could be measured after exposure to clonidine. Attenuation of cAMP levels was also seen following co-expression of the dopamine D2 receptor along with dopamine administration. Thus, after inducing a receptor-mediated tone in adenylyl cyclase activity, Xenopus oocytes can be conveniently used to study also ligands that bind to inhibitory G-protein coupled receptors.

The kinetics of competitive radioligand binding predicted by the law of mass action.

Although equilibrium competitive radioligand binding studies are often used to characterize hormone and neurotransmitter receptors, the kinetics of such experiments have not been extensively explored. The interactions of the radioligand and competitor with the receptors can be described by two differential equations which can be solved to yield a single equation describing the binding of the radioligand as a function of time. This equation has several applications: First, it can be used to simulate competitive binding reactions under defined conditions. Second, fitting experimental data to this equation allows one to determine the association and dissociation rate constants of the competing ligand, parameters that cannot be derived from equilibrium experiments. Furthermore, this method can be used to determine the KI of the competing drug from data acquired before equilibrium is reached. Third, mathematical analysis of the binding equation allowed us to answer two specific questions regarding the kinetics of competitive radioligand binding: how long such an incubation takes to equilibrate, and how the IC50 varies over time. The answers to these questions depended, to a large extent, on the relative values of the dissociation rate constants of the radioligand and competitor, which can be determined as noted above. When the competitor dissociates from the receptors more rapidly than the radioligand, the IC50 first decreases and then increases, but never has a value less than the KI. At low radioligand concentrations, equilibrium is reached in the same amount of time required of the radioligand to dissociate completely from the receptors as determined in an "off-rate experiment." At higher concentrations of radioligand this time is halved. When the competitor dissociates from the receptor more slowly than does the radioligand, then the time required to equilibrate depends only on the dissociation rate constant of the competitor, and the IC50 decreases over time.

Use of superoxide dismutase and catalase to protect catecholamines from oxidation in tissue culture studies.

A new enzymatic approach for the prevention of catecholamine oxidation that is particularly useful for studies with cultured cells is described. Catecholamine oxidation was assayed by acid alumina chromatography using a modified procedure that yields greater than or equal to 90% column recovery of catecholamine concentrations as low as 1.0 nM. Addition of superoxide dismutase and catalase (10-25 micrograms/ml each) results in virtually complete inhibition of catecholamine oxidation under a variety of experimental conditions. Although superoxide dismutase could prevent catecholamine oxidation, addition of catalase helped prevent the cytotoxicity of oxidative products. When used together, these enzymes have no effect on cell growth, hormonal response, or radioligand binding to membrane beta-adrenergic receptors in the murine S49 lymphoma cell, a widely used model system for studying catecholamine action. Combined use of superoxide dismutase and catalase offers nonperturbing, long-lasting protection of catecholamines in studies with cells in vitro. This method provides a useful alternative to ascorbic acid, chelators, or reducing agents which have previously been used to prevent catecholamine oxidation but which may have other effects on cultured cells and on membrane proteins.

Functional expression of B2 bradykinin receptors from Balb/c cell mRNA in Xenopus oocytes.

The murine BALB/c 3T3 fibroblast clone SV-T2 (3T3 cells) expresses receptors for the nonapeptide bradykinin. In these cells, bradykinin stimulates both inositol phosphate (InsP) formation and arachidonic acid release by independently activating phospholipase C and phospholipase A2, respectively. These actions of bradykinin are mediated by a receptor(s) coupled to pertussis toxin-insensitive guanine nucleotide-binding proteins. Bradykinin-stimulated increases in InsP lead to the mobilization of intracellular Ca2+. We examined the expression of 3T3 receptors for bradykinin in oocytes from Xenopus laevis, cells capable of in vitro expression of foreign mRNA for receptors coupled to the mobilization of Ca2+. Poly(A)+ mRNA was prepared from 3T3 cells and expression of receptors for bradykinin was demonstrated by agonist-mediated stimulation of 45Ca2+ efflux from oocytes injected with 50 ng of poly(A)+ RNA. Bradykinin-stimulated efflux of 45Ca2+ was dose dependent (EC50 = 15 nM) and blocked by the specific mixed B1,B2 bradykinin antagonist NPC 567 but not by the B1 antagonist desArg9[Leu8]bradykinin. Size fractionation of 3T3 poly(A)+ RNA on a sucrose gradient demonstrated a single peak of bradykinin-stimulated 45Ca2+ efflux, with an approximate mRNA size of 4.5 kilobases. Bradykinin-stimulated 45Ca2+ efflux in size-fractionated mRNA was clearly separable from response to [Arg]vasopressin at another receptor linked to InsP formation and Ca2+ mobilization in 3T3 cells.

Expression of beta-adrenergic receptors in synchronous and asynchronous S49 lymphoma cells. I. Receptor metabolism after irreversible blockade of receptors and in cells traversing the cell division cycle.

We have examined the metabolism of beta-adrenergic receptors in intact S49 lymphoma cells. Centrifugal elutriation was used to prepare synchronized cells enriched in particular phases of the cell division cycle. In these synchronized cells, the rate of appearance of beta-adrenergic receptors (i.e., [125I]iodocyanopindolol binding sites) was continuous, approximately 75 sites/cell/hr, and receptor number per cell increased in proportion to the increase in cell size. Thus, receptor number, normalized for cell size, remains constant throughout the cell cycle. Receptors on cells in G1, S, and G2/M phases of the cell cycle displayed similar affinities for the radiolabeled antagonist [125I]iodocyanopindolol and apparent affinities for the agonist isoproterenol. We examined rates of receptor turnover in asynchronous cells by following receptor recovery after inactivation of beta-adrenergic receptors with bromoacetylalprenololmenthane (BAAM), an irreversible beta-adrenergic antagonist. The beta-adrenergic receptors on S49 cells demonstrated an average "half-life" of 28-30 hr. Since the population doubling time for S49 cells is 16-17 hr, this would indicate that receptor protein is conserved through successive cell generations. Moreover, receptor reappearance after blockade by BAAM was a function of newly appearing receptors during the S49 cell cycle and not loss of BAAM from receptors. The rate of receptor metabolism indicates that, under basal conditions (i.e., in the absence of agonist), beta-adrenergic receptors on S49 cells are metabolized more slowly than are other classes of receptors that bind peptides and cholinergic agonists in several other cell types.

Expression of beta-adrenergic receptors in synchronous and asynchronous S49 lymphoma cells. II. Relationship between receptor number and response.

We have used two experimental approaches--receptor inactivation with an irreversible antagonist and changes in receptor expression during passage of cells through the cell cycle--to explore the relationship between beta-adrenergic receptor number and response in intact S49 lymphoma cells. beta-Receptors in asynchronous cultures of S49 cells were blocked to varying degrees with the irreversible antagonist bromoacetylalprenololmenthane (BAAM). Blockade by BAAM was noncompetitive and did not alter the affinity of receptors for the agonist isoproterenol. Intracellular accumulation of cAMP in response to 1 microM isoproterenol was proportional to receptor number both at times of initial and maximal accumulation. In contrast, when intracellular accumulation of cAMP in response to isoproterenol was measured in synchronized cultures of S49 cells (obtained by centrifugal elutriation), a notably different relationship was observed. Cells were least responsive, that is, receptors appeared "uncoupled," during S phase of the cell cycle. This attenuation of response was not due to alterations of receptor number, receptor affinity for agonist, or expression of the catalytic unit of adenylate cyclase. Use of the antibiotic mycophenolic acid, a selective inhibitor of the synthesis of GTP, elicited response patterns in asynchronous cells similar to those seen in synchronized cells. These results confirm that wild-type S49 cells do not possess spare receptors. In addition to the importance of total receptor number in determining maximal response to isoproterenol, receptors may show differential efficacy in promoting cAMP accumulation as cells traverse the cell cycle. Changes in cellular levels or utilization of GTP during the cell cycle may serve to regulate the coupling of receptors to the stimulation of adenylate cyclase.

Certain beta-blockers can decrease beta-adrenergic receptor number: II. Down-regulation of receptor number by alprenolol and propranolol in cultured lymphoma and muscle cells.

We have used two different cultured cell lines--S49 lymphoma cells and BC3H-1 muscle cells--to examine the regulation of beta-adrenergic receptors by receptor antagonists. Rather than an increase ("up-regulation") of receptor number that such antagonists often produce, we found that certain beta-blockers elicit a decrease ("down-regulation") of beta-adrenergic receptors. Alprenolol and propranolol, but not sotalol or ICI 118,551, at concentrations of 10-100 nM down-regulated beta-adrenergic receptors 20-70% following 16-20 hours of treatment of S49 or BC3H-1 cells. Several observations suggest that this phenomenon depends upon beta-receptor interaction, including stereoselectivity [(-)-enantiomers more potent than (+)-enantiomers], blockade of the effect by ICI 118,551, absence of down-regulation of alpha-adrenergic receptors in BC3H-1 cells, and lack of a decrease in beta-adrenergic receptor-independent (forskolin-stimulated) cyclic AMP accumulation in S49 cells. The possibility of retained antagonist interfering with receptor measurement was precluded by the fact that the antagonist-induced decrease in receptor number required several hours incubation and occurred without a prominent change in receptor affinity. The ability of the beta-blockers to elicit down-regulation did not correlate with hydrophobicity of the drugs. Antagonist-induced down regulation of beta-adrenergic receptors did not occur in S49 lymphoma cells that lack the alpha-subunit of Gs, the guanine nucleotide-binding regulatory protein, thus implying a requirement for receptor-alpha s interaction in eliciting beta-receptor down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic analysis of beta-adrenergic receptor internalization and down-regulation.

We have used wild-type and variants of the T-lymphoma cell line S49 to explore internalization and down-regulation of adenylate cyclase-linked beta-adrenergic receptors. Internalization was defined by the loss in "surface receptors" detected at 4 degrees C on intact cells by the antagonists [3H]CGP-12177 or [125I]iodocyanopindolol, whereas down-regulation was defined as the loss in total cellular content of receptors [( 125I]iodocyanopindolol binding assayed at 37 degrees C). In wild-type cells, the beta-adrenergic agonist isoproterenol induced a rapid (t 1/2, approximately equal to 1 min) and reversible loss in surface receptors. The surface sites were lost at a rate similar to the rate of desensitization of beta-adrenergic receptor-mediated cyclic AMP generation of S49 cells. A series of S49 variants (cyc-, UNC, H21a) having lesions in NS (the guanine nucleotide binding protein that couples beta-receptors to adenylate cyclase) or with absent cAMP-dependent protein kinase activity (kin-), had a loss in surface sites that was equivalent to that of wild-type cells. By contrast, S49 variant cells having lesions in NS showed variable rates and extents of down-regulation of beta-adrenergic receptors. In wild-type and kin- S49 cells, beta-receptors down-regulated with a t 1/2 of approximately equal to 4 hr. Down-regulation was blunted in the cyc- and UNC variants that have altered coupling of receptors to NS, but it was faster in the H21a variant that retains receptor-NS interaction. Recovery of receptors after down-regulation occurred at a similar rate (t 1/2, approximately equal to 6 hr) in wild-type, UNC, and H21a cells. These results demonstrate that internalization of beta-adrenergic receptors may be necessary, but is not sufficient, to explain agonist-induced receptor down-regulation in S49 cells. The variable expression in the development of down-regulation in S49 variants implies that receptor-NS interaction regulates the fate of receptors linked to the stimulation of adenylate cyclase.

Do agonists promote rapid internalization of beta-adrenergic receptors?

We used elution of radioligands at low pH to quantitate intracellular beta-adrenergic receptors on intact S49 lymphoma cells. We validated this method with respect to cell viability, beta-adrenergic receptor integrity, and transferrin receptors on these cells. On control cells, about 15% of the radiolabeled beta-adrenergic antagonists [3H]dihydroalprenolol and [125I]iodocyanopindolol specifically bound at 37 degrees C could not be eluted at low pH; these binding sites appear to be intracellular receptors that are inaccessible to the surface-restricted antagonist [3H]CGP-12177 [tritiated (+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazole-2-one hydrochloride]. Incubation of cells with the agonist isoproterenol at 37 degrees C for 15 min did not change the number of [3H]dihydroalprenolol binding sites but reduced [3H]CGP-12177 binding sites by 50% or more. However, all specifically bound [3H]CGP-12177 and [3H]dihydroalprenolol were eluted by acid. In addition, the number of acid-elution-resistant [125I]iodocyanopindolol binding sites was not increased in cells coincubated with 1 microM isoproterenol and [125I]iodocyanopindolol for 15 min at 37 degrees C, even though those sites show a loss in apparent affinity for isoproterenol of about 2 orders of magnitude, a loss previously attributed to internalization. We conclude that the early phase of agonist-mediated desensitization of beta-adrenergic receptors in S49 cells does not coincide with the movement of receptors to intracellular sites; instead, agonist-modified receptors remain in association with the plasma membrane and are accessible to the extracellular environment. These "redistributed" receptors together with "cell-surface" and "intracellular" receptors represent three classes of beta-adrenergic receptors that can be selectively identified in intact target cells.

Anti-Rho(D) IgG binds to band 3 glycoprotein of the human erythrocyte membrane.

Alkali-extracted erythrocyte ghost membranes from Rho(D)-positive and Rho(D)-negative donors were incubated with human immune anti-Rho(D) IgG and nonimmune IgG. After sensitization with IgG, the integral membrane proteins were solubilized in Brij 36T nonionic detergent and chromatographed by gel filtration. There was a distinct resolution of IgG into free and membrane-complexed forms. The IgG-complexed membrane proteins were isolated by the use of a staphylococcal protein A affinity support. The protein A-bound complexes were examined for polypeptide composition by gel electrophoresis after elution. Only Rho(D)-positive membrane proteins incubated with immune anti-Rho(D) IgG revealed intact band 3. Control Rh-negative membrane proteins that had reacted with immune anti-Rho(D) IgG and the Rh-positive membranes that had reacted with nonimmune IgG showed only low molecular weight fragments of band 3 that bound nonspecifically to IgG. Arguments are presented supporting a band 3 localization for the Rh antigen.