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The C terminus of the human V2 vasopressin receptor contains multiple phosphorylation sites including a cluster of amino acids that when phosphorylated prevents the return of the internalized receptor to the cell surface. To identify the step where the recycling process was interrupted, the trafficking of the V2 receptor was compared with that of the recycling V1a receptor after exposure to ligand. Initially, both receptors internalized in small peripheral endosomes, but a physical separation of their endocytic pathways was subsequently detected. The V1a receptor remained evenly distributed throughout the cytosol, whereas the V2 receptor accumulated in a large aggregation of vesicles in the proximity of the nucleus where it colocalized with the transferrin receptor and Rab11, a small GTP-binding protein that is concentrated in the perinuclear recycling compartment; only marginal colocalization of Rab11 with the V1a receptor was observed. Thus, the V2 receptor was sequestered in the perinuclear recycling compartment. Targeting to the perinuclear recycling compartment was determined by the receptor subtype and not by the inability to recycle, since the mutation S363A in the phosphorylation-dependent retention signal generated a V2 receptor that was recycled via the same compartment. The perinuclear recycling compartment was enriched in beta-arrestin after internalization of either wild type V2 receptor or its recycling mutant, indicating that long term interaction between the receptors and arrestin was not responsible for the intracellular retention. Thus, the fully phosphorylated retention domain overrides the natural tendency of the V2 receptor to recycle and, by preventing its exit from the perinuclear recycling compartment, interrupts its transit via the "long cycle." The data suggest that the inactivation of the domain, possibly by dephosphorylation, triggers the return of the receptor from the perinuclear compartment to the plasma membrane.

The human V2 vasopressin receptor contains one consensus site for N-linked glycosylation at asparagine 22 in the predicted extracellular amino terminal segment of the protein. This segment also contains clusters of serines and threonines that are potential sites for O-glycosylation. Mutagenesis of asparagine 22 to glutamine abolished N-linked glycosylation of the V2 receptor (N22Q-V2R), without altering its function or level of expression. The N22Q-V2R expressed in transfected cells migrated in denaturing acrylamide gels as two protein bands with a difference of 7000 Da. Protein labeling experiments demonstrated that the faster band could be chase to the slower one suggesting the presence of O-linked sugars. Sialidase treatment of membranes from cells expressing the N22Q-V2R or of immunoprecipitated metabolically labeled V2R accelerated the migration of the protein in acrylamide gels demonstrating the existence of O-glycosylation, the first time this type of glycosylation has been found in a G protein coupled receptor. Synthesis of metabolically labeled receptor in the presence of 1 mM phenyl-N-acetyl-alpha-D-galactosaminide, a competitive inhibitor of N-acetyl-alpha-D-galactose and N-acetylneuraminic acid transferases, also produced a receptor that migrated faster in denaturing gels. Serines and threonines present in the amino terminus were analyzed by alanine scanning mutagenesis to identify the acceptor sites. O-glycosylation was found at most serines and threonines present in the amino terminus. Because the disappearance of a site opened the availability of others to the transferases, the exact identification of the acceptor sites was not feasible. The wild type V2R expressed in HEK 293, COS, or MDCK cells underwent N- and O-linked glycosylation. The mutant V2R bearing all serine/threonine substitutions by alanine at the amino terminus yielded a receptor functionally indistinguishable from the wild type protein, whose mobility in polyacrylamide gels was no longer affected by sialidase treatment.

We reported recently the molecular cloning of a genomic fragment, designated S31, that has an open reading frame of 1095 nucleotides, encoding a protein of 365 amino acids. Amino acid similarity analysis suggested that the S31 protein could be a guanine nucleotide-binding protein-coupled receptor pertaining to the serotonin receptor subfamily. Expression of the S31 open reading frame in murine L cells confirmed this, because it led to the appearance of serotonin-mediated inhibition of adenylyl cyclase activity, which was absent in the recipient L cells. We now report some aspects of the pharmacological profile of this receptor. We found that the relative potencies with which 5-hydroxytryptamine, 5-carboxamidotryptamine, methysergide, ergotamine, 8-hydroxydipropylaminotetralin, and trifluoromethylphenylpiperazine promote inhibition of adenylyl cyclase are as follows: 5-hydroxytryptamine >> methysergide >> ergotamine >> trifluoromethylphenylpiperazine > or = 8-hydroxydipropylaminotetralin > 5-carboxyamidotryptamine. This corresponds to the rank order of potencies assigned for these drugs for the 1E subtype of serotonin receptors discovered by Leonhardt and collaborators in human brain [J. Neurochem. 53:465-471 (1989)].

We investigated the biochemical and functional properties of five vasopressin V2 receptor mutants (L44F, L44P, W164S, S167L, and S167T) that were recently described in families with a history of X-linked nephrogenic diabetes insipidus. COS.M6 cells transfected with cDNA encoding these mutants acquired < 4% specific [3H]arginine vasopressin (AVP) binding sites on the cell surface in comparison with cells transfected with cDNA coding for the wild-type receptor. Membrane preparations from COS.M6 cells or human embryonic kidney 293 cells expressing these mutants did not respond with an increase in adenylyl cyclase activity in response to AVP, which is in contrast to membranes from cells expressing the wild-type. By analyzing fusion proteins of the V2 receptor and Escherichia coli alkaline phosphatase attached to the carboxyl terminus of the receptor moiety, we found that the mutants L44P, W164S, S167L, and S167T lacked complex glycosylation and were expressed at low levels. The data suggest that the mutants L44P, W164S, S167T, and S167L are misfolded and therefore retained within the endoplasmic reticulum and degraded. In contrast, the fusion proteins carrying the mutant L44F and the in vitro mutant S167A were expressed in their mature form at wild-type levels; however, only the mutant S167A was functionally active. Site-directed mutagenesis of S167 revealed that elimination of the endogenous hydroxyl group (S167A) yielded a protein with properties identical to those of the wild-type receptor, whereas both the introduction of a methyl group (S167T) and the replacement of the hydroxyl group by an isopropyl group (S167L) profoundly disturbed receptor processing. The data show that minute changes at codon 167 nearly abolish expression of a mature protein, thus defining structural requirements of this codon.

The human V2 vasopressin receptor belongs to the superfamily of G protein-coupled receptors believed to be anchored to the plasma membrane by seven transmembrane regions. The extracellular portion of the human V2 vasopressin receptor contains one site susceptible to N-linked glycosylation. Metabolic labeling and immunoprecipitation of the receptor expressed in transfected cells were applied to examine whether the protein was indeed glycosylated. The V2 vasopressin receptor expressed transiently was glycosylated, but glycosidase treatment to test the complexity of the sugar moiety linked to asparagine revealed that the majority of the receptor protein lacked complex carbohydrates, an indication of an improperly processed protein. This immature protein displayed a tendency to form aggregates. In contrast with these data, testing of the sugar complexity of the receptor protein synthesized in stably transfected cells identified the predominant form as an appropriately processed receptor protein. Mutagenesis of asparagine 22 to glutamine produced on expression in transfected cells a nonglycosylated receptor with ligand binding affinity and coupling characteristics almost identical to those of the wild-type form. After exposure to elevated concentrations of AVP (100 nM), the nonglycosylated form desensitized to the same extent as the wild-type receptor.

Cholecystokinin (CCK)-A and CCK-B receptors are highly homologous members of the seven transmembrane domain G-protein-coupled receptor superfamily. Genes of both receptors contain five exons and share a similar exon-intron organization. To determine the structural basis of CCK-A receptor (CCK-AR) functionally coupled to Gs, a series of chimeric mutants were constructed by replacing exons of human CCK-B receptor (CCK-BR), from the second to the fifth (last) exon, with human CCK-AR counterparts. Binding and signal transduction properties of wild-type and chimeric receptors were examined in stably transfected HEK-293 cells. Chimeric receptors that maintained high affinity binding to CCK exhibited dose-dependent increases in intracellular calcium mobilization similar to both wild-type receptors. However, only the wild-type CCK-AR and chimeric mutants containing the second exon of CCK-AR were able to mediate significantly greater increases in intracellular cAMP content and adenylyl cyclase activity compared with wild-type CCK-BR. A CCK-BR mutant was further constructed by replacing five amino acids, Gly-Leu-Ser-Arg-(Arg)-Leu, in the first intracellular loop with the corresponding five CCK-AR specific amino acids, Ile-Arg-Asn-Lys-(Arg)-Met. The resultant receptor maintained high affinity binding to both CCK and gastrin and dose-dependent calcium responses similar to wild-type CCK-BR. However, this first intracellular loop mutant also gained positive cAMP responses to both sulfated CCK-8 and gastrin-17 with EC50 values of 8.5 +/- 1 nM and 23 +/- 7 nM, respectively. These data suggest that the first intracellular loop of CCK-AR is essential for coupling to Gs and activation of adenylyl cyclase signal transduction cascade.

Chick oviduct progesterone-receptor proteins were treated in cytosol with the reversible cross-linking reagent methyl 4-mercaptobutyrimidate. The product of the reaction was a 7S complex that could be detected and recovered after sucrose-density-gradient centrifugation in 0.3M-KCl. The extent of the reaction was dependent on the concentration of methyl 4-mercaptobutyrimidate and independent of the presence of bound hormone, since unlabelled receptors could also be cross-linked. The cross-linking reaction required conditions in which the cytosol 6S complex was preserved. A Stokes radius of 7.3 nm was determined by gel filtration in Agarose A-1.5 m in 0.3 M-KCl. The sedimentation coefficient, which was also determined in 0.3 M-KCl, allowed us to calculate a mol. wt. of 228,000. We were also able to cross-link partially purified receptor forms isolated by using an Agarose A-15 m column. On reduction with beta-mercaptoethanol the complex broke down to 4S monomers that were identified by DEAE-cellulose and phosphocellulose chromatography, adsorption on DNA-cellulose and gel filtration in an Agarose A-1.5 m column. In most cases, A and B receptor proteins were released in equivalent amounts, implying that the cross-linked form was an A-B complex.

Several reports have appeared recently which contradict our earlier findings on the identity and characterization of chick progesterone receptor. Puri et al. (Puri, R. K., Grandics, P., Dougherty, J. J., and Toft, D. O. (1982) J. Biol. Chem. 257, 10831-10837) have isolated a protein with a molecular weight of Mr approximately 88,000 and have reported that this protein is the “nontransformed” molybdate-stabilized receptor. Because of this discrepancy from our own work, we have repeated their methods and obtained purified samples of this protein. In this paper we show that the Mr = 88,000 +/- 2,000 protein is not a receptor but is apparently an artifact of the use of particular steroid affinity resins. Evidence presented to show that this protein is not the authentic receptor include: 1) it is obtained from affinity resins in 10-fold molar excess over the mass of authentic receptor; 2) its tryptic map does not resemble either authentic receptor subunit; 3) it can be resolved from authentic hormone-binding receptors on DEAE-Sephadex A-25 chromatography; 4) the protein does not bind progesterone; and finally 5) its elution from affinity matrices is not biospecific for progesterone and can even be eluted in equivalent yields with buffer alone. We conclude that the Mr = 88,000 protein is not a progesterone-binding protein.

A cDNA of 2149 base pairs with an incomplete open reading frame (ORF) encoding amino acids 1–516 of a 531-amino acid protein highly homologous to muscarinic receptors was cloned from a rat brain cDNA library. The complete ORF was then deduced from a DNA fragment cloned from a rat genomic library. This ORF was subcloned into the eukaryotic expression vector p91023(B) under control of the adenovirus major late promoter and co-transfected with the thymidine kinase selection marker into muscarinic receptor-negative, thymidine kinase-negative murine L cells. Stable transformants were selected and tested for acquisition of muscarinic receptors by following appearance of specific binding sites for the muscarinic ligand [3H] N-methylscopolamine. Two cell lines, LM5.36 and LM5.40, were cloned and shown to express typical muscarinic receptor sites, thus confirming that the newly cloned ORF encodes a muscarinic receptor, the rat M5 muscarinic acetylcholine receptor. Tests for activities showed it to stimulate phosphoinositide hydrolysis in intact cells, without affecting positively or negatively adenylyl cyclase activity. The M5 receptor contains two putative glycosylation sites at its amino terminus and, based on hydropathicity analysis, is predicted to span the plasma membrane seven times. Like 17 other receptors of this class, the M5 receptor has 19 conserved amino acids, among which are 4 prolines located in the 4th, 5th, 6th, and 7th predicted transmembrane regions, conferring possible bends to these helices, and 2 cysteines, one in the 1st and the other in the 2nd extracellular loop, possibly providing for a disulfide bond. Similarity in amino acid composition and in patterns of antagonist binding and biologic effects suggest the M5 receptor to be M1-like.

Partial proteolytic fragmentation of the two chick oviduct progesterone receptor subunits was used to identify structural features shared by the two proteins. Both subunits can be photoaffinity labeled at their hormone-binding sites (Birnbaumer, M., Schrader, W. T., and O'Malley, B. W. (1983) J. Biol. Chem. 258, 1637-1644) using the radioactive steroid [methyl-3H] 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione. Native subunits A (Mr = 79,000) and B (Mr = 108,000) were partially purified, photoaffinity-labeled, and then subjected to various mild proteolytic digestions. Labeled fragments were analyzed by fluorography after electrophoresis of the digests under denaturing conditions. Digestion patterns were characteristic for each protease tested. However, fragments from both A and B were indistinguishable for all peptides of less than Mr = 60,000. Time course studies demonstrated the sequential production of progressively smaller discrete fragments in a manner consistent with a precursor-product relationship among them and established the existence of similar structural domains resistant to proteolysis in both proteins. Autoradiographic peptide maps were obtained by 125I-labeling of pure A and B protein isolated by two-dimensional gel electrophoresis followed by exhaustive tryptic digestion and two-dimensional separation. These studies revealed that a significant proportion of the smaller A protein differs in its primary sequence from that of the B protein which excludes the possibility of their sharing a precursor-product relationship. We conclude that B and A subunits are separate proteins with common structural features in the native state, but with considerable amino acid sequence differences. The simplest hypothesis consistent with these findings is that B and A are the products of two separate genes which have diverged to give rise to two different but related proteins that fold in such a manner as to be almost indistinguishable by proteolytic attack of their native conformation.

Many hormones have been shown to activate phospholipase C, which results in the hydrolysis of membrane polyphosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PIP2). Two second messengers are known to be produced by PIP2 hydrolysis, 1,2-diacylglycerol, an endogenous activator of a family of enzymes called protein kinase C (PKCs), and inositol 1,4,5-trisphosphate, which raises free levels of intracellular Ca2+. Treatment of various cells with 4 beta-phorbol 12-myristate 13-acetate (PMA), a specific exogenous activator of PKCs, causes an enhancement or sensitization of adenylyl cyclase activities. This finding prompted us to examine the effects of direct hormonal activation of PIP2 hydrolysis on the sensitization of adenylyl cyclase. Liao et al. [J. Biol. Chem. 265:11273-11284 (1990)] have shown that P2 purinergic receptor agonists such as ATP and muscarinic receptor agonists such as carbachol stimulate PIP2 hydrolysis in L cells expressing the M5 muscarinic acetylcholine receptor. We investigated the effects of these hormones on adenylyl cyclase and contrasted these effects with the sensitizing effects of PMA. We found that ATP pretreatment of two different types of L cells resulted in a rapid 50-150% sensitization of prostaglandin E1-, epinephrine-, and forskolin-stimulated adenylyl cyclase activity, with an EC50 of 3 microM ATP. This effect was qualitatively similar to that caused by 10 nM PMA. The enhancement of adenylyl cyclase activity was associated with an increase in the Vmax for hormonal stimulation and with a lack of significant effects of ATP on the EC50. The effect was completely eliminated when adenylyl cyclase was assayed in the presence of high free Mg2+ levels (10 mM). Down-regulation of PKCs with long term PMA treatment did not affect the ATP-induced sensitization of adenylyl cyclase, although the PMA-induced sensitization of adenylyl cyclase was eliminated. In contrast to the effects of ATP and PMA, treatment of the cells with carbachol alone had no effect on adenylyl cyclase; however, in combination with nanomolar concentrations of PMA, synergism of the sensitization of adenylyl cyclase was observed. These data indicate that the activation of P2 purinergic receptors by ATP, and possibly activation of M5 muscarinic receptors by carbachol, may be important in the signal transduction pathways leading to the increases in the responsiveness of hormone-stimulated adenylyl cyclase.

The V1a arginine vasopressin receptor (V1aR) expressed in HEK 293 cells was phosphorylated after binding to arginine vasopressin (AVP). The phosphate was incorporated very rapidly into the protein but remained attached for a very short time despite the continuous presence of hormone. The extent of phosphorylation depended upon the concentration of AVP suggesting the involvement of G-protein-coupled receptor kinases. Protein kinase C (PKC) contributed to V1aR phosphorylation as demonstrated by the fact that inhibition of the kinase decreased the amount of phosphate incorporated into the receptor. However, PKC activity was not responsible for the transient nature of V1aR phosphorylation. The hormone-free receptor could be phosphorylated by phorbol ester-activated PKC. Although the phosphorylation was transient, the phosphate groups incorporated remained on the receptor protein longer than those incorporated after AVP treatment. PKC phosphorylation of unoccupied V1aR was not sufficient to promote sequestration. Vasopressin also promoted sequestration of about 80% of the surface receptor, but measurements of the rate of accumulation of inositol phosphates in the sustained presence of the ligand did not reveal a significant desensitization of coupling to phospholipase C activity. The addition of a V1aR antagonist inhibited the sustained accumulation of inositol phosphates establishing that the sustained stimulation of PLC was mediated by receptors located on the cell surface. The transient character of V1aR phosphorylation seemed intrinsic to the receptor protein rather than a consequence of signaling within the cell, and receptor sequestration appeared to be responsible for the desensitization observed in HEK 293 cells.

The V2 vasopressin receptor undergoes ligand-induced sequestration and desensitization (Birnbaumer, M., Antaramian, A., Themmen, A. P. N., and Gilbert, S. (1992) J. Biol. Chem. 267, 11783-11788). The V2 receptor expressed in transfected cells labeled with [32P] orthophosphate was phosphorylated following the addition of 100 nM arginine vasopressin (AVP). Phosphorylation was complete 5 min after addition of AVP, and was not stimulated by increased levels of Ca2+ or cAMP. The half-maximal dose of AVP that stimulated phosphorylation was 2.4 +/- 0.4 nM, similar to the receptor KD of 4. 5 +/- 0.4 nM. The role of phosphorylation on receptor desensitization was investigated by studying two vasopressin receptors 14 and 27 amino acids shorter than the wild type receptor. The missing segments were not needed for normal ligand binding or coupling to Gs, but the last 14 amino acids were required for phosphorylation. The truncated receptors exposed to 100 nM AVP were sequestered and desensitized. The R137H V2R mutant receptor that binds vasopressin with wild type-like affinity and does not couple to Gs (Rosenthal, W., Antaramian, A., Gilbert, S., and Birnbaumer, M. (1993) J. Biol. Chem. 268, 13030-13033) was phosphorylated and subjected to ligand-induced sequestration. These results established that phosphorylation is not essential for sequestration and desensitization of the V2 vasopressin receptor. Furthermore, they revealed that the conformation acquired after ligand occupancy is necessary for receptor phosphorylation and sequestration, while coupling to Gs is not.

Methods were developed to adequately extract, separate and, without the use of NaF as stabilizing agent, purify to better than 90% purity human erythrocyte Ns and Ni, the stimulatory and inhibitory guanine nucleotide- and Mg-binding regulatory components of adenylyl cyclases, as well as a protein containing Mr = 35,000 subunits. On the basis of a functional assay for Ns, it was purified about 5,000-fold from starting washed erythrocyte membranes with a yield of about 10%. A typical purification yields from 60 units of outdated human blood, between 500 and 1,000 micrograms of pure Ns, and a similar amount of Ni. Pure Ns and Ni contain each at least one alpha and one beta subunit (Northup, J.K., Sternweis, P.C., Smigel, M.D., Schleifer, L.S., Ross, E.M., and Gilman, A.G. (1980) Proc. Natl. Acad. Sci. U.S.A. 74, 6516-6520; Codina, J., Hildebrandt, J.D., Iyengar, R., Birnbaumer, L., Sekura, R.D., and Manclark, C.R. (1983) Proc. Natl. Acad. Sci. U.S.A. 77, 4276-4280). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at varying acrylamide concentrations yielded Mr values of 42,000 and 40,000 for the alpha subunits of Ns and Ni, and of 35,000 for the beta subunits of Ns and Ni. Two-dimensional thin layer analysis of tryptic peptides obtained from digesting 125I-labeled subunits of Ns and Ni confirmed the finding of Manning, D., and Gilman, A.G. (1983) J. Biol. Chem. 258, 7059-7063) that while their alpha subunits are clearly different, their beta subunits are the same. Hydrodynamic analysis of the molecular weights of the nondenatured proteins showed behavior consistent with Mr = 95,500 for Ns, the same for Ni, and Mr = 40,000 for the protein containing the Mr = 35,000 beta subunit. Sedimentation coefficients and Stokes radii of the purified Ns were indistinguishable from those of Ns activity present in initial cholate extracts from human erythrocyte membranes. Further, the overall kinetics with which Ns activity in cholate extracts and Ns activity in the purified protein reconstituted the Ns-deficient adenylyl cyclase system of cyc- S49 cells was also indistinguishable. We conclude that we have purified the native unactivated form of Ns, and by serendipity the Ni, as well as a protein containing the 35 kDa beta subunit of Ns and Ni.(ABSTRACT TRUNCATED AT 400 WORDS)

Chick progesterone receptor subunits A and B have been photoaffinity-labeled using [3H]R5020 ([17 alpha-methyl-3H]17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione) by a modification of the procedures previously reported by our laboratory (Dure, L. S., IV, Schrader, W. T., and O'Malley, B. W. (1980) Nature (Lond.) 283, 784-786). [3H]R5020 binds to the same receptor sites as authentic progesterone, and has an apparent Kdiss of 2.0 nM. Use of a CuSO4 filter raised the coupling efficiency to 5% and labeled exclusively the receptor proteins. Smaller labeled macromolecules were found to be proteolytic fragments of receptors. The protease(s) could not be inhibited by any of the commonly used protease inhibitors. However, the proteolytic activity was completely removed by passage of crude receptor preparations through phosphocellulose columns. Receptor preparations, photoaffinity-labeled after this procedure, showed exclusively one radioactive band at Mr = 79,000 (subunit A) or Mr = 108,000 (subunit B) with no detectable side-reaction products. Labeled receptors A and B were digested with Staphylococcus aureus V8 protease to yield smaller [3H]R5020-protein fragments derived from both. Molecular weight estimates (Mr = 9,500) and apparent isoelectric points indicate similarities of these regions of both A and B. The photoaffinity protocol described here thus provides a method for study of the hormone-binding domain of progesterone receptors and of receptor proteolysis in crude extracts.

We studied the relative ability to activate phospholipase C (PLC) of four Gs-coupled receptors expressed in L cells at different densities. Stable cell lines expressing various levels of the luteinizing hormone receptor (LHR), the type 2 vasopressin receptor (V2R), or the type 1 or type 2 beta-adrenergic receptor (beta 1- or beta 2AR) were isolated. The PLC activity was assessed by the measurement of free intracellular Ca2+ concentrations and the accumulation of inositol phosphates. We previously reported that, at 24,000 sites/cell, the LHR in L cells stimulated adenylyl cyclase by 10-fold over basal levels and PLC by 50% over basal levels. The EC50 for stimulation was 20-fold higher for PLC than for adenylyl cyclase. We now report that LHR tends to stimulate PLC more at a higher receptor density and less at a lower density. EC50 values for accumulation of inositol phosphates remained unchanged. The human V2R and the human beta ARs are strong adenylyl cyclase stimulators, and their potential for dual signaling was unknown. Expressing the V2R at 100,000 sites/cell or more and the beta ARs at 300,000 sites/cell resulted in stimulation of PLC by these receptors. As with the LHR, higher concentrations of vasopressin or isoproterenol were needed to reach 50% stimulation of PLC, compared with that of adenylyl cyclase. The beta 1AR was a stronger PLC stimulator than was the beta 2AR. The orders of potency for isoproterenol, epinephrine, and norepinephrine to stimulate adenylyl cyclase and PLC were the same for each of the two beta ARs. These results indicate that the ability of Gs-coupled receptors to stimulate PLC is dependent on the levels of receptor expression, and they suggest that dual signaling potential is a common property of Gs-coupled receptors and possibly also of G(i)-coupled receptors.

1. The structure of human plasma low-density lipoprotein(s) [LD lipoprotein(s)] was investigated by several immunological and optical techniques. The effects of delipidation and of chemical modification by 3-carboxypropionylation, acetylation, diazotization and amidination were examined. A sensitive double-antibody radioimmunoassay for human LD lipoproteins is presented and is used to assess the extent of immunochemical modification. A computer best-fit analysis is used to analyse circular-dichorism (c.d.) spectra. These methods permit comparisons of the relative effects of chemical modification and delipidation of LD lipoprotein under similar experimental conditions. 2. 3-Carboxypropionylation, acetylation or diazotization produces qualitative and quantitative changes in the immunochemical properties of LD lipoprotein. Amidination causes minor changes detected by radioimmunoassay but not by double-diffusion experiments. In general, the order of effectiveness in displacing 125I-labelled LD lipoprotein is amidinated>diazo or acetyl>3-carboxypropionyl derivatives. 3. The order of the extent of conformational alteration induced in apoLD lipoprotein and LD lipoprotein, as judged by c.d. analyses, was 3-carboxypropionylation>diazotization>acetylation or amidination. 4. Delipidation of LD lipoprotein results in immunological alterations that are qualitatively detected by antisera to LD lipoprotein. Four of five antisera to apoLD lipoprotein form precipitin lines of identity between native LD lipoprotein and apoLD lipoprotein in double-diffusion experiments. An anti-(apoLD lipoprotein) serum that forms precipitin lines of complete identity between LD lipoprotein and apoLD lipoprotein reacts differently with these two antigens in radioimmunoassay. ApoLD lipoprotein is only one-fourth to one-half as effective as LD lipoprotein, on a protein basis, in the displacement of 125I-labelled LD lipoprotein from this anti-(apoLD lipoprotein). 5. Conformational analysis indicates that apoLD lipoprotein retains a high proportion of the structural integrity of the native lipoprotein. Delipidation induces a small decrease in the content of β-structure and a small increase in disordered structure, without greatly affecting the α-helical content. Chemical modification produces more severe conformational changes of apoLD lipoprotein than of LD lipoprotein. Computer analysis of the c.d. spectra of apoLD lipoprotein indicates that addition of high concentrations of sodium decyl sulphate abolishes most of the β-conformation concomitant with increases in α-helical and disordered structure. 6. There is parallelism between the alteration of the charge of LD lipoprotein and apoLD lipoprotein and the extent of immunochemical and conformational changes.