Prestin topology: localization of protein epitopes in relation to the plasma membrane.

Computer modeling of the outer hair cell (OHC) motor protein prestin produces ambiguous results regarding transmembrane regions and localization of its termini. To determine the location of prestin's N- and C-termini, we created prestin constructs with synthetic epitopes located immediately upstream or downstream of prestin. The spatial distribution of these epitopes was studied in prestin-transfected cells using immunofluorescence. In permeabilized cells, antibodies label the plasma membrane of 30% of the cells, reflecting transfec- tion efficiency. Under non-permeabilizing conditions, the few labeled cells also displayed a lack of plasma membrane integrity. These data suggest that prestin's N-and C-termini are cytoplasmic. Furthermore, prestin staining in OHCs was observed only under permeabilizing conditions. These results implicate prestin's N- and C-termini as portions that may interact with other cytoplasmic proteins. A model of prestin membrane topology is also considered based on the results.

Mutual up-regulation of thyroid hormone and parathyroid hormone receptors in rat osteoblastic osteosarcoma 17/2.8 cells.

PTH and thyroid hormone (T(3)) stimulate anabolic and catabolic processes in bone predominantly by acting on osteoblasts. Both inadequate and excessive secretion of either hormone can result in clinical bone disorders. In addition, T(3) and PTH related peptide (PTHrP) have multiple effects on a wide number of other tissues modulating both cell differentiation and proliferation. To address the question of whether there might be functional mutual regulation of T(3) receptors (TR) and PTH/PTHrP receptors (PTHR), we studied their expression and receptor-mediated intracellular effects in rat osteoblastic osteosarcoma (ROS) 17/2.8 cells. PTHR were up-regulated by T(3) pretreatment (10(-)(10)-10(-)(6) M) in ROS 17/2.8 cells in a dose-dependent manner. T(3) pretreatment increased both PTH-induced cyclic AMP response element binding protein (CREB) phosphorylation and PTH-induced intracellular calcium transients, and further decreased PTH-induced down-regulation of alkaline phosphatase activity, suggesting that there are functional consequences of the PTHR up- regulation. Pretreatment with PTH (10(-)(10)-10(-)(6) M) or PTHrP (10(-)(9) M) for 3-4 days resulted in a dose-dependent up-regulation of TR in ROS 17/2.8 cells. cAMP analogues or a calcium ionophore were able to mimic the effect of PTH on TR binding, suggesting that either the cAMP-signaling pathway or Ca(2+) could be involved in PTH-induced up-regulation of the TR. These observations provide a novel example of mutual interactions between nuclear receptors and membrane receptors and may have significant implications for the regulation of bone remodeling in health and disease.

Prestin is the motor protein of cochlear outer hair cells.

The outer and inner hair cells of the mammalian cochlea perform different functions. In response to changes in membrane potential, the cylindrical outer hair cell rapidly alters its length and stiffness. These mechanical changes, driven by putative molecular motors, are assumed to produce amplification of vibrations in the cochlea that are transduced by inner hair cells. Here we have identified an abundant complementary DNA from a gene, designated Prestin, which is specifically expressed in outer hair cells. Regions of the encoded protein show moderate sequence similarity to pendrin and related sulphate/anion transport proteins. Voltage-induced shape changes can be elicited in cultured human kidney cells that express prestin. The mechanical response of outer hair cells to voltage change is accompanied by a 'gating current', which is manifested as nonlinear capacitance. We also demonstrate this nonlinear capacitance in transfected kidney cells. We conclude that prestin is the motor protein of the cochlear outer hair cell.

Insulin-like growth factor I production is essential for anabolic effects of thyroid hormone in osteoblasts.

Thyroid hormone (T3) and insulin-like growth factor I (IGF-I) are critical regulators of skeletal function. T3 increases IGF-I production in bone. To assess the potential role of IGF-I as a mediator of T3 actions, we characterized phenotypic markers of osteoblast activity in two osteoblast models, normal mouse osteoblasts and MC3T3-E1 cells, exposed to T3 alone or under conditions that interfere with IGF-I actions. T3 significantly increased osteoblast 3H-proline incorporation, alkaline phosphatase (ALP), and osteocalcin. Both alphaIR3, a neutralizing monoclonal antibody to the IGF-I receptor, and JB1, an IGF-I analogue antagonist, attenuated the stimulatory effects of T3. T3 effects also were decreased in cells transfected with antisense oligonucleotide (AS-ODN) to the IGF-I receptor gene. Both IGF-I and T3 had mitogenic effects that were inhibited by the antagonists. IGF-I by itself did not stimulate 3H-proline incorporation, ALP, and osteocalcin in the models used, revealing that although IGF-I is essential for the anabolic effects of T3, it acts in concert with other factors to elicit these phenotypic responses.

Nuclear receptor corepressors activate rather than suppress basal transcription of genes that are negatively regulated by thyroid hormone.

A group of transcriptional cofactors referred to as corepressors (CoRs) were recently shown to play a central role in basal silencing of genes that contain positive triiodothyronine (T3) response elements. In a reciprocal manner, negatively regulated genes are stimulated by unliganded thyroid hormone receptor (TR) and repressed upon the addition of T3. We used a TR beta mutant, called P214R, which fails to interact with CoRs, to examine whether CoRs also play a role in the control of genes that are negatively regulated in response to T3. In studies of three negatively regulated genes (the pituitary thyroid-stimulating hormone alpha-subunit [TSH alpha], TSH beta, and hypothalamic thyrotropin-releasing hormone [TRH] genes), stimulation of basal promoter activity by unliganded TR beta was impaired by introducing the P214R CoR mutation. Coexpression of each of the CoRs SMRT (silencing mediator for retinoid receptors and TRs) and NCoR (nuclear receptor CoR) enhanced basal stimulation of the negatively regulated promoters in a TR-dependent manner, but this effect was not seen with the P214R TR mutant. The mechanism of CoR effects on negatively regulated promoters was explored further with a series of GAL4-TR chimeric receptors and mutants that allowed TR effects to be assessed independently of receptor interactions with DNA. These experiments revealed that, like the negative regulation of genes by wild-type TR, basal activation occurred with GAL4-TR, but not with the GAL4-P214R mutant, and was reversed by the addition of T3. These results suggest that TR interactions with negatively regulated genes may be driven through protein-protein interactions. We conclude that a subset of negatively regulated genes are controlled by a novel mechanism that involves TR-mediated recruitment and basal activation by SMRT and NCoR. Addition of T3 reverses basal activation, perhaps by dissociation of CoRs.

Thyroid hormone (T3) inhibits ciprofibrate-induced transcription of genes encoding beta-oxidation enzymes: cross talk between peroxisome proliferator and T3 signaling pathways.

Peroxisome proliferators cause rapid and coordinated transcriptional activation of genes encoding peroxisomal beta-oxidation system enzymes by activating peroxisome proliferator-activated receptor (PPAR) isoform(s). Since the thyroid hormone (T3; 3,3',5-triiodothyronine) receptor (TR), another member of the nuclear hormone receptor superfamily, regulates a subset of fatty acid metabolism genes shared with PPAR, we examined the possibility of interplay between peroxisome proliferator and T3 signaling pathways. T3 inhibited ciprofibrate-induced luciferase activity as well as the endogenous peroxisomal beta-oxidation enzymes in transgenic mice carrying a 3.2-kb 5'-flanking region of the rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene fused to the coding region of luciferase. Transfection assays in hepatoma H4-II-E-C3 and CV-1 cells indicated that this inhibition is mediated by TR in a ligand-dependent fashion. Gel shift assays revealed that modulation of PPAR action by TR occurs through titration of limiting amounts of retinoid X receptor (RXR) required for PPAR activation. Increasing amounts of RXR partially reversed the inhibition in a reciprocal manner; PPAR also inhibited TR activation. Results with heterodimerization-deficient TR and PPAR mutants further confirmed that interaction between PPAR and TR signaling systems is indirect. These results suggest that a convergence of the peroxisome proliferator and T3 signaling pathways occurs through their common interaction with the heterodimeric partner RXR.

The thyrotropin (TSH) receptor transmembrane domain mutation (Pro556-Leu) in the hypothyroid hyt/hyt mouse results in plasma membrane targeting but defective TSH binding.

The hyt/hyt mouse is hypothyroid because of a mutation in the TSH receptor (TSH-R). In this report, we confirm the presence of a Pro to Leu mutation in amino acid 556 of the fourth transmembrane domain (TM4) of the TSH-R. This Pro is highly conserved in members of the G protein-coupled seven-transmembrane family of receptors. Insertion of this mutation into the wild-type rat receptor eliminated TSH binding and receptor function in transfected 293 and COS cells. Wild-type TSH-R conferred a 7.4-fold increase in cAMP and a 2.3-fold stimulation of a cAMP-responsive reporter gene. The P556L mutant receptor elicited no increase in cAMP or the reporter gene. Cells transfected with wild-type receptor bound TSH with a Kd of 3.3 x 10(-10) M, whereas no TSH binding was detected with the P556L mutant. Because the P556L mutation occurs in a receptor region (TM4) that is not expected to alter the binding of TSH, additional studies were performed to examine receptor processing and cellular localization. Mutant receptors from solubilized membranes also failed to bind TSH, indicating that the absence of binding to intact cells was not accounted for intracellular trapping of the mutant receptor. Western blot analyses demonstrated that the mutant and wild-type receptors were processed through a similar series of precursors and that a mature 95-kilodalton form of the mutant TSH-R was produced, consistent with its insertion into the plasma membrane. Immunofluorescence studies confirmed expression of the P556L mutant on the cell surface of transfected cells and in thyroid tissue from hyt/hyt mice. Although the extracellular domain of the TSH-R is sufficient for high affinity binding of TSH, we conclude that the hyt mutation in the fourth transmembrane domain eliminates TSH binding. These results suggest interactions between the extracellular and transmembrane domains of the TSH-R and indicate that this highly conserved proline is required for normal receptor structure and function.

Negative regulation of the glycoprotein hormone alpha gene promoter by thyroid hormone: mutagenesis of a proximal receptor binding site preserves transcriptional repression.

Transcription of the glycoprotein hormone alpha gene is repressed by the thyroid hormone receptor (TR) in a hormone dependent manner. Previous studies identified a TR binding site immediately downstream of the TATA box. Site directed mutagenesis and transient gene expression studies were used to evaluate the role of this TR binding site as a negative thyroid response element (nTRE). Mutagenesis of the putative negative thyroid response element (nTRE) site eliminated TR binding but failed to eliminate negative regulation by T3. A mutation which converted the putative nTRE to a higher affinity palindromic element did not enhance repression, but rather eliminated thyroid hormone dependent negative regulation. Proximal alpha promoter sequences between -100 and +44 were replaced with a heterologous thymidine kinase promoter resulting in a construct that was not repressed by T3 treatment. This finding confirmed that repression required proximal alpha promoter sequences and also indicated that repression did not occur by interference with the function of upstream the alpha gene enhancers. These studies indicate that TR binding adjacent to the TATA box is not required for T3 mediated repression of the alpha promoter and suggest that negative regulation may involve protein-protein interactions with promoter-specific transcription factors.

Localization of promoter sequences required for thyrotropin-releasing hormone and thyroid hormone responsiveness of the glycoprotein hormone alpha-gene in primary cultures of rat pituitary cells.

The glycoprotein hormone alpha-gene is regulated by multiple hormones in different pituitary and placental cell types. In thyrotropes, the alpha-gene is stimulated by TRH and repressed by thyroid hormone (T3). We used transient expression assays in primary cultures of rat pituitary cells to examine regulation of the alpha-promoter (alpha Luc) by TRH and T3. The -846 alpha Luc activity was stimulated 3.4-fold by TRH and repressed 44% by T3. GnRH and cAMP stimulated -846 alpha Luc by 8.3- and 8.6-fold, respectively. T3 blocked TRH stimulation, but it had no effect on stimulation by GnRH or cAMP, suggesting that the T3-mediated effects are thyrotrope specific. TRH and T3 responsiveness was preserved with deletions to -346 basepairs (bp). TRH responsiveness was lost after deletion to -280 bp, whereas T3-mediated repression was eliminated by further deletion to -180 bp. A series of DNA fragments between -420 and -180 was linked to -132 alpha Luc to study TRH and T3 responses in greater detail. Sequences between -346 to -180 bp conferred TRH responsiveness and T3 inhibition. TRH responsiveness was not seen after 3'-deletions of this fragment to -244 or -280 bp. These results together with the 5'-deletions provide evidence for two interdependent TRH regulatory regions: one between -346 to -280 bp and another between -244 to -180 bp. T3-dependent repression only requires sequences between -244 and -180 bp. Site-directed cluster mutations were created in each of these two regulatory domains. A mutation in region 1 (-346 to -328 bp) eliminated TRH stimulation, but retained basal suppression by T3.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid hormone receptor mutants that cause resistance to thyroid hormone. Evidence for receptor competition for DNA sequences in target genes.

Several distinct mutations in the ligand-binding domain of the beta form of the thyroid hormone receptor have been reported in kindreds with the autosomal dominant syndrome of generalized resistance to thyroid hormone (GRTH). GRTH receptor mutants are functionally inactive but capable of inhibiting normal receptor function in transient expression studies. We examined the possibility that this dominant negative activity of the GRTH mutants involves competition for receptor binding to DNA. Mutations introduced into either the T3 ligand-binding domain (LBD) or into the DNA-binding domain (DBD) of the receptor eliminated the transcriptional activity of the receptor. In cotransfection experiments, the LBD mutants, but not the DBD mutants, inhibited the transcriptional activity of the normal receptor. The inhibitory activity of the LBD mutants was abolished by the introduction of an additional mutation into the DBD, suggesting that the DBD is required for dominant negative activity. A chimeric receptor, in which the DNA-binding domain of the thyroid hormone receptor was exchanged with the homologous region in the glucocorticoid receptor (GTG), was used to study thyroid hormone receptor competition for GTG interactions with thyroid receptor target sequences. In the absence of thyroid hormone, the normal thyroid hormone receptor inhibited dexamethasone stimulated transcription by GTG. The transcriptional activity of GTG was also inhibited by the LBD mutants but not by a DBD mutant of the thyroid hormone receptor. These results indicate that the thyroid hormone receptor mutations that occur in GRTH compete with normal receptors at DNA-binding sites in target genes to block normal receptor function.

Thyroid hormone receptor mediates transcriptional activation and repression of different promoters in vitro.

The thyroid hormone receptor (TR) has the dual ability to activate or repress transcription of specific genes. A cell-free transcription system was used to study the effects of TR on transcription by positively (TREpMLP) and negatively (TSH alpha) regulated promoters. Receptor-deficient HeLa cell extracts were complemented with baculovirus-produced TR. TR stimulated transcription from the TREpMLP promoter by 3-fold, and trans-activation did not require hormone. Transcriptional stimulation by TR required the presence of the TRE sequence and was diminished by the addition of competitor TRE binding sites. Baculovirus-produced TR repressed transcription in vitro from the TSH alpha promoter by 30-50%, also in a hormone-independent manner. Transcription from a control adenovirus 2 major late promoter was unaffected by added TR. Receptor-specific antisera and competition with TRE binding sites impaired TR-mediated repression of the TSH alpha promoter. Unlike transcriptional stimulation, which was optimal when TR and HeLa extracts were added concomitantly, transcriptional repression by the TR was most effective when the receptor was preincubated with the alpha-promoter, suggesting that receptor binding to the promoter may block access of other proteins to cause transcriptional repression. These results indicate that baculovirus-expressed TR mediates transcriptional activation and repression in a promoter-specific manner in vitro. This system provides a valuable model for examining transcriptional control by the TR.

Thyroid hormone resistance syndrome. Inhibition of normal receptor function by mutant thyroid hormone receptors.

Thyroid hormone (T3) resistance is inherited in most cases in an autosomal dominant manner. The disorder is characterized by elevated free thyroid hormone levels and partial resistance to thyroid hormone at the cellular level. Distinct single amino acid substitutions in the ligand binding domain of the beta form of the thyroid hormone receptor have been described in two kindreds with this disorder. We used transient expression assays to characterize the functional properties of these receptor mutants, one containing a Gly to Arg change at amino acid 340 (G340R) and the other a Pro to His change at amino acid 448 (P448H). A nine amino acid carboxy terminal deletion (delta 448-456), analogous to an alteration that occurs in v-erbA, was also studied for comparison with the mutations that occur in the T3 resistance syndrome. None of the receptor mutants were able to mediate thyroid hormone dependent activation (TreTKCAT) or repression (TSH alpha CAT) of reporter genes when compared with the wild type receptor. In addition, the mutants inhibited the activity of normal alpha and beta receptor isoforms when examined in coexpression assays. This activity, referred to as dominant negative inhibition, was manifest with respect to both the positively and negatively regulated reporter genes. Although mutant receptor binding to DNA was unaffected, ligand binding studies showed that the G340R and delta 448-456 mutants failed to bind T3, whereas the P448H mutant bound hormone with reduced affinity (approximately 10% of normal) compared to the wild type receptor. Consistent with this finding, the P448H mutant receptor was partially active at higher T3 concentrations. Furthermore, the dominant negative inhibition elicited by the P448H receptor mutant at higher T3 concentrations was reversed in the presence of high doses of T3. These findings indicate that mutant beta receptors in patients with thyroid hormone resistance have reduced affinity for T3 and are functionally deficient, but impair the activity of normal receptors, thereby providing a mechanism for the dominant mode of inheritance in this disorder.

Human thyroid hormone beta 1 receptor produced by recombinant baculovirus-infected insect cells.

Thyroid hormone receptors are nuclear proteins which regulate transcription in a hormone dependent manner. The baculovirus expression system was used for the overexpression of the beta 1 isoform of the human thyroid hormone receptor. The baculovirus produced receptor binds tri-iodothyronine with high affinity, is specifically immunoprecipitated with a beta 1 specific antibody, and binds to DNA that contains a known thyroid hormone receptor recognition site. Large scale production and purification of baculovirus produced receptor will be useful for structure-function analyses and studies of transcriptional regulation.

Repression of the human glycoprotein hormone alpha-subunit gene by glucocorticoids: evidence for receptor interactions with limiting transcriptional activators.

Expression of the glycoprotein hormone alpha gene is regulated divergently by glucocorticoids in different cell types. Coexpression of the glucocorticoid receptor (GR) with an alpha-CAT reporter gene caused activation of alpha promoter activity in fibroblasts, but repression in JEG-3 choriocarcinoma cells, indicating that cell-specific factors dictate positive vs. negative regulation of this promoter by GR. Cell-specific sequences and other enhancer elements in the the alpha gene have been relatively well characterized in JEG-3 cells, and this model was used to further examine the mechanism of transcriptional repression by glucocorticoids. Promoter mutagenesis indicated that the degree of GR-mediated repression was impaired by a variety of deletional and site-directed mutations between -171 and -111 bp, a region that includes both cell-specific and cAMP response elements (CREs). In an attempt to further localize a negative glucocorticoid response element (GRE) sequence, binding studies were used to assess GR interactions with alpha promoter DNA sequences. Using avidin-biotin complex DNA binding assays, a series of overlapping alpha promoter DNA sequences between -170 to 29 basepairs were tested, but each failed to bind GR, whereas a control GRE avidly bound receptor. Similarly, in competition assays in transfected CV-1 cells, the alpha gene 5'-flanking sequence did not compete for GR stimulation of a glucocorticoid responsive reporter gene, whereas a sequence that contains known GR-binding sites (murine mammary tumor virus) effectively inhibited GR-mediated expression. The absence of high affinity GR-binding sites in the alpha promoter suggested that mutations that affected GR inhibition may have eliminated recognition sites for transactivators, which are themselves targets for the GR, rather than altering specific negative GRE sites in the DNA sequence. To examine this possibility, GR repression was studied using chimeric transcription factors. The transcription-activating domains of several different proteins (CREB, thyroid hormone receptor, or VP16) were linked to the DNA-binding domain of Gal-4, and transcription was driven by the Gal-4 recognition site (UAS). GR markedly repressed transactivation by Gal-4-CREB and, to a lesser degree, the Gal-4-thyroid hormone receptor and Gal-4-VP16 chimeric proteins. Repression occurred when UAS was linked to either the alpha promoter or to the E1B promoter. Thus, inhibition occurs in the absence of either the CRE or the proximal alpha promoter. These results support a mechanism in which GR-mediated repression in JEG-3 cells occurs by receptor interference with the transactivating potential of enhancer-binding proteins or associated transcription factors.

Negative and positive transcriptional regulation by thyroid hormone receptor isoforms.

Multiple forms of human thyroid hormone (T3) receptor have been identified, including true receptors that bind T3 (alpha 1 and beta) and a splicing variant (alpha 2) that does not bind T3. The alpha 1- and beta-receptors activate transcription through interactions with positive thyroid response elements (TREs). The alpha 2 variant is unable to activate transcription and has been reported to inhibit alpha 1 or beta stimulation of positive TREs, a property referred to as dominant negative activity. In this report we have performed studies to assess the functional properties of different members of the thyroid receptor family with regard to both positive and negative transcriptional regulation. The alpha 1-, alpha 2-, and beta-receptors were each coexpressed in JEG-3 cells with either TreTKCAT (CAT = chloramphenicol acetyltransferase), a reporter gene that contains a positive TRE, or TSH alpha CAT, a negatively regulated reporter gene. The alpha 1 and beta isoforms stimulated transcription of TreTKCAT and inhibited TSH alpha CAT transcription in a T3-dependent manner, whereas the alpha 2 variant was inactive. When coexpressed with alpha 1- or beta-receptors, alpha 2 inhibited regulation of positive TREs, but the effects of alpha 2 were modest and only occurred when relatively high doses of receptor were transfected. The alpha 2-receptor variant did not affect negative regulation by alpha 1- or beta-receptors. Thus, in both positive and negative regulation, thyroid hormone receptor isoforms that bind T3 (alpha 1, beta) are functional, whereas the alpha 2 isoform, which does not bind T3, is not functional.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of cholecystokinin receptors in pancreas.

Specific labeling of a major Mr 85-95 K protein was obtained using the SH, NH2 heterobifunctional cross-linker m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS) to affinity label cholecystokinin (CCK) receptors on rat pancreatic plasma membranes, pancreatic acinar cells and acinar cell tumor membranes with 125I-CCK-33. Endoglycosidase F (endo F) digestion of this species in gel slices indicated that at least two components were present which contain N-linked glycans. The smaller protein of Mr approximately 85 K was digested by endo F to a final product of approximately Mr 62 K while the larger Mr approximately 95 K protein generated two endo F products of Mr 55 K and Mr 43 K. These findings suggest that the receptor for CCK on pancreatic acinar cells exhibits an oligomeric structure, possessing two distinct CCK-binding proteins.

Use of the heterobifunctional cross-linker m-maleimidobenzoyl N-hydroxysuccinimide ester to affinity label cholecystokinin binding proteins on rat pancreatic plasma membranes.

The binding of 125I-cholecystokinin-33 (125I-CCK-33) to its receptors on rat pancreatic membranes was decreased by modification of membrane protein sulfhydryl groups. Sulfhydryl modifying reagents also caused an accelerated release of bound 125I-CCK-33 from its receptor. Because of the presence of an essential sulfhydryl group(s) in CCK receptor binding we studied the application of the heterobifunctional (SH,NH2) cross-linker, m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), to affinity label 125I-CCK-33 binding proteins on rat pancreatic plasma membranes. Analysis of the cross-linked products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that this heterobifunctional cross-linker affinity labeled a major Mr = 80,000-95,000 protein previously identified as part of the CCK receptor on the basis of affinity labeling using homobifunctional and heterobifunctional photoreactive cross-linkers. Additional proteins of Mr greater than 200,000, and Mr = 130,000-140,000 were affinity labeled using MBS. The efficiency of the cross-linking reaction between 125I-CCK-33 and its membrane binding proteins with MBS was significantly greater than that obtained with NH2-directed homobifunctional reagents such as disuccinimidyl suberate. The efficiency of cross-linking could be dramatically improved by reduction of membrane proteins with low-molecular weight thiols prior to binding and cross-linking. The differential labeling patterns of the CCK binding proteins obtained with chemical cross-linkers of similar length but different chemical reactivity underscores the need for caution in predicting native receptor structure from affinity labeling data alone. Using the same pancreatic plasma membrane preparation and 125I-insulin, the Mr = 125,000 alpha-subunit of the insulin receptor was affinity labeled using MBS as cross-linker, demonstrating its utility in identifying other peptide hormone receptors.

Analysis of cholecystokinin-binding proteins using endo-beta-N-acetylglucosaminidase F.

We have previously shown that the cholecystokinin (CCK)-binding proteins in rat pancreatic plasma membranes consist of a major Mr 85,000 and minor Mr 55,000 and Mr 130,000 species as revealed by affinity labeling with 125I-CCK-33 using the cross-linker, disuccinimidyl suberate. The glycoprotein nature of these species was investigated using endoglycosidase F (endo F) and neuraminidase treatment and wheat germ agglutinin-agarose chromatography. Treatment of affinity-labeled membranes with endo F resulted in increased electrophoretic mobilities of all three binding proteins, indicating removal of N-linked oligosaccharide side chains. Endo F treatment of each protein in gel slices indicated the following cleavage relationships: Mr 85,000----65,000; Mr 55,000----45,000; Mr 130,000----110,000. Using limiting enzyme conditions to digest each protein contained in excised SDS gel slices, three and four products, respectively, were identified for the Mr 85,000 and 55,000 proteins. Similar treatment of the Mr 130,000 protein revealed only the Mr 110,000 product. These results indicated that the Mr 85,000 protein has at least three, the Mr 55,000 protein has at least four, and the Mr 130,000 protein has at least one, N-linked oligosaccharide side chain(s) on their polypeptide backbone. Neuraminidase treatment of affinity-labeled membranes caused slight increases in the electrophoretic mobilities of all three proteins, indicating the presence of sialic acid residues. Solubilization of affinity-labeled membranes in Nonidet P-40 followed by affinity chromatography on wheat germ agglutinin-agarose revealed that all three CCK-binding proteins specifically interact with this lectin and can be eluted with N-acetyl-D-glucosamine. Analysis of the proteins present in the eluted fractions by silver staining indicated a significant enrichment for proteins having molecular weights corresponding to the major CCK-binding proteins in comparison to the pattern of native membranes. Taken together, these studies provide definitive evidence that the CCK-binding proteins in rat pancreas are (sialo)glycoproteins.

Regulation of surface topography of mouse peritoneal cells. Formation of microvilli and vesiculated pits on omental mesothelial cells by serum and other proteins.

The mesothelial cells of the mouse omentum provide an in vivo model for the study of the mobilization of labile microvilli on the cell surface. These mesothelial cells are sparsely covered with microvilli and large pits 150--400 nm in diameter, termed vesiculated pits. On the unstimulated cell, the microvilli average 44/100 microns2 and pits, 30/100 microns 2 of surface and they are rapidly induced to increase in number by the intraperitoneal injection of isologous mouse serum. After 2 min, microvilli increase threefold, continue to sevenfold at 30 min, and decrease to fourfold at 90 min. Vesiculated pits increased with similar kinetics. Bovine serum albumin and gamma globulin also stimulate the microvilli and pits to form, but the response is a slow, gradual rise to five- or sixfold the normal value at 90 min. Evidence indicates that multiple factors, possibly including insulin and immunoglobulins, are involved in the effect of serum. The close physical and temporal relationship between microvilli and pits suggests that a correlation exists in their mobilization by the cell and it is hypothesized that microvilli function in the regulation of the cortical microfilament network in effecting this mobilization.