Biomedical subjects

B F Roettger

Publications and source records attributed to B F Roettger.

10 recordsLinked to original sources

PubMed1999

Regulation of lateral mobility and cellular trafficking of the CCK receptor by a partial agonist.

Partial agonists are effective tools for advancing development of highly selective drugs and providing insights into molecular regulation of cellular functions. Here, we explore the impact of a partial agonist on key aspects of cholecystokinin (CCK) receptor regulation, its lateral mobility and cellular trafficking, in native pancreatic acinar cells and Chinese hamster ovary cells expressing CCK receptor (CHO-CCKR). We developed and characterized a novel fluorescent partial agonist, rhodamine-Gly-[(Nle28, 31)CCK-26-32]-phenethyl ester, that binds specifically and with high affinity to CCK receptors. Such analogs are fully efficacious pancreatic acinar cell secretagogues without supramaximal inhibition that mobilize intracellular calcium with little or no increase in phospholipase C (PLC) activity. Despite minimal phosphorylation of CCK receptors in response to this partial agonist, receptor trafficking was the same as that observed with full agonist (CCK). This included normal internalization via clathrin-dependent endocytosis in CHO-CCKR cells and insulation on the surface of pancreatic acinar cells. Also, as with CCK-occupied receptor, fluorescence recovery after photobleaching of partial agonist-occupied receptor on the acinar cell surface demonstrated a marked temperature-dependent slowing of its rate of diffusion. This was similarly associated with resistance to acid-induced dissociation of ligand. Thus some key molecular regulatory mechanisms for CCK receptor internalization and insulation may be initiated by cellular signaling cascades that are not dependent on PLC activation or receptor phosphorylation.

Animals↗

PubMed1997

Quantitative dynamic multicompartmental analysis of cholecystokinin receptor movement in a living cell using dual fluorophores and reconstruction of confocal images.

Receptor regulation is a key component of the phenomenon of desensitization in response to agonist stimulation which protects cells from overstimulation. Receptor internalization is one part of this response, often quantified by the portion of saturable ligand binding which becomes resistant to acidic washes. It is now clear that this can include receptor in multiple distinct cellular compartments. We have developed a morphological technique involving dual fluorescent probes to delineate the plasmalemma and the ligand-occupied receptor using confocal microscopy, with analysis involving three-dimensional reconstruction and quantitation of receptor movement through each compartment. When a radioiodinated cholecystokinin (CCK) analogue occupied its receptor on the CHO-CCKR cell line, it became progressively more resistant to dissociation with acidic medium. Quantitation of receptor internalization in these cells over time using this dynamic morphological technique correlated with the acid-resistant receptor fraction, and provided the additional information of the cellular compartments traversed. This technique will have multiple applications to explore the cell-specific handling of this and other ligand-occupied receptors.

Animals↗

PubMed1997

Cholecystokinin-induced desensitization, receptor phosphorylation, and internalization in the CHP212 neuroblastoma cell line.

Agonist stimulation of cells often results in desensitization of the response, to protect the cell from overstimulation. We have previously shown that the type A cholecystokinin (CCK) receptor on the pancreatic acinar cell and on the model CHO-CCKR cell line undergoes desensitization in response to CCK, with receptor phosphorylation and internalization playing key roles. Although these mechanisms contribute in a cell-specific manner, no analogous information exists for the CCK receptor expressed on neuronal cells, where in vivo data demonstrate a particularly sensitive response to CCK. The present study was designed to explore CCK receptor desensitization in the CHP212 neuroblastoma cell line, focusing on inositol 1,4,5-trisphosphate (IP3) responses to CCK and on recognized molecular and cellular mechanisms of desensitization. CCK promptly stimulated IP3 responses in these cells, with hormonal responsiveness rapidly and completely desensitized. Both receptor phosphorylation and internalization were observed to occur, with the former occurring most rapidly and likely being responsible for the earliest desensitization observed. Although the time course of receptor phosphorylation and dephosphorylation, and the groups of kinases involved in the neuroblastoma cell line, were most similar to those in the pancreatic cell, the movement of the agonist-bound receptor in these cells was quite different from that in the pancreatic cell and most similar to that in the CHO-CCKR cell line. This hybrid response supports the cell-specific nature of CCK receptor regulation and provides an important system to explore the molecular determinants of these processes.

Dose-Response Relationship, Drug↗

PubMed1997

Roles of cholecystokinin receptor phosphorylation in agonist-stimulated desensitization of pancreatic acinar cells and receptor-bearing Chinese hamster ovary cholecystokinin receptor cells.

Receptor phosphorylation has been implicated in desensitization responses to some agonist ligands, in which receptors may become uncoupled from G proteins and move into cellular compartments inaccessible to hydrophilic ligands. Understanding of the linkage between these processes, however, has come largely from recombinant receptor-bearing cell systems with consensus sites of kinase action mutagenized. We recently established methodology permitting direct assessment of sites of phosphorylation of the cholecystokinin receptor (CCKR) in its native milieu in the pancreatic acinar cell and in a Chinese hamster ovary (CHO)-CCKR cell line (1, 2). Although CCK binding leads to phosphorylation of serine residues within the third intracellular loop of the receptor in both cell types, there are clear differences in the time course of phosphorylation, in the balance of action of kinases and a receptor phosphatase, and in a few of the distinct sites phosphorylated. In this work, we have directly assessed the inositol 1,4,5-triphosphate responses to CCK and desensitization of these responses in both cells. CHO cell lines expressing receptor mutants with protein kinase C consensus sites modified were also studied. CCK-stimulated inositol 1,4,5-triphosphate responses in both cells expressing wild-type receptors were rapidly and completely desensitized, associated with the onset of receptor phosphorylation. However, despite maintenance of the phosphorylated state of the receptor in the CHO-CCKR cell and its dephosphorylation returning the receptor to its basal state in the acinar cell, desensitization continued to be present in both. Mutagenesis of Ser260 and Ser264 to alanines individually reduced receptor phosphorylation by approximately 50%, whereas the dual mutant completely eliminated agonist-stimulated phosphorylation. Because other sites of phosphorylation were still intact in this construct, this raises the possibility of hierarchical phosphorylation with these two sites key in making other sites accessible to kinases. Constructs modifying Ser264 delayed the onset of desensitization, whereas all constructs proceeded to achieve complete desensitization by 10 min. Receptor internalization occurred independent of its phosphorylation state in the CHO cell lines, explaining the desensitization observed. In the acinar cell in which the receptor remains on the cell surface after agonist occupation, we postulate that receptor insulation achieves similar uncoupling from G protein association as is achieved by receptor phosphorylation early after agonist occupation.

Animals↗

PubMed1997

Cellular handling of unoccupied and agonist-stimulated cholecystokinin receptor determined by immunolocalization.

Cellular handling of receptor molecules is an important mechanism for the regulation of appropriately sensitive hormone-stimulated signaling. Until now, our understanding of the cellular handling of the cholecystokinin (CCK) receptor has been largely limited to following a tagged ligand through the cell. In the present work, we report the application of unique CCK receptor antisera directed toward intracellular domains, which permitted the immunolocalization of this molecule independently of its occupation with ligand. The CCK receptor antisera were also useful in Western blotting and immunoprecipitation of this receptor. Unstimulated CCK receptors remained on the surface of both recombinant stable rat CCK-A receptor-bearing Chinese hamster ovary cell line (CHO-CCKR) cells and native rat pancreatic acinar cells and did not constitutively internalize. Agonist stimulation of the CHO-CCKR cells resulted in the prompt internalization of a subset of surface receptors, representing those that were occupied with ligand. Unoccupied receptors remained on the surface, uninfluenced by the stimulated signaling pathways. Consistent with this, CCK receptor phosphorylation induced by 12-O-tetradecanoylphorbol-13-acetate treatment did not stimulate receptor internalization. After internalization, we observed substantial receptor recycling to the plasma membrane. These insights provide the first evidence that CCK receptor internalization occurs as a direct result of an induced conformational change and presumed bimolecular interaction, rather than as an effect of a signaling event.

Amino Acid Sequence↗

PubMed1997

Antagonist-stimulated internalization of the G protein-coupled cholecystokinin receptor.

Receptor-mediated endocytosis has been observed after agonist occupation of several G protein-coupled receptors, which contributes to the desensitization response to agonist stimulation; however, the cellular signals required to initiate this process are unclear. In this study, we developed and characterized a new antagonist analogue of cholecystokinin (D-Tyr-Gly-[(Nle28,31,D-Trp30)cholecystokinin-26-32]-phen eth yl ester) that can be tagged with a fluorescent rhodamine and radioiodinated. This has permitted us to demonstrate that antagonist occupation of the cholecystokinin receptor also results in receptor internalization, which dissociates this response from second messenger signaling activities and receptor phosphorylation. Immunolocalization of this receptor after occupation with an established nonpeptidyl antagonist confirmed this phenomenon. Antagonist-induced receptor internalization probably results from stabilization of the receptor in a conformation that exposes a domain critical to directing it into the clathrin-dependent endocytic pathway. This work provides evidence for a new and independent mechanism for receptor internalization, provides a mechanism for the rarely observed phenomenon of antagonist-induced desensitization, and raises important issues regarding the approach to establish optimal treatment regimens for antagonist drugs.

Binding, Competitive↗

PubMed1996

Role of receptor phosphorylation in desensitization and internalization of the secretin receptor.

The secretin receptor is prototypic of a recently described family of G protein-coupled receptors. We recently demonstrated its phosphorylation in response to agonist stimulation and elimination of this covalent modification by C-terminal truncation (F. Ozcelebi et al. (1995) Mol. Pharmacol. 48, 818-824). Here, we explore the functional impact of receptor phosphorylation and structural determinants for desensitization by comparing receptor behavior after agonist exposure in cell lines expressing wild-type and truncated receptor. To characterize receptor internalization, a novel fluorescent full agonist, [rat secretin-27]-Gly-rhodamine, was developed, which bound specifically and with high affinity. Both receptor constructs bound secretin normally, leading to normal G protein coupling and cAMP accumulation and prompt receptor internalization. Exposure to 10 nM secretin for 5 min or 12 h prior to washing and restimulation with a full range of concentrations demonstrated absent cAMP responses in wild-type receptor-bearing cells and responses 25 to 30% of control and shifted 1 order of magnitude to the right in the truncated receptor-bearing cells. Thus, the major mechanism of desensitization was phosphorylation-independent receptor internalization. Phosphorylation was associated with a distinct process that likely represents interference with G protein coupling, manifest as a reduced rate of cAMP stimulation. Thus, dual distinct mechanisms of desensitization exist in the secretin receptor family that should help protect receptor-bearing cells from overstimulation.

Animals↗

PubMed1995

Dual pathways of internalization of the cholecystokinin receptor.

Receptor molecules play a major role in the desensitization of agonist-stimulated cellular responses. For G protein-coupled receptors, rapid desensitization occurs via receptor phosphorylation, sequestration, and internalization, yet the cellular compartments in which these events occur and their interrelationships are unclear. In this work, we focus on the cholecystokinin (CCK) receptor, which has been well characterized with respect to phosphorylation. We have used novel fluorescent and electron-dense CCK receptor ligands and an antibody to probe receptor localization in a CCK receptor-bearing CHO cell line. In the unstimulated state, receptors were diffusely distributed over the plasmalemma. Agonist occupation stimulated endocytosis via both clathrin-dependent and independent pathways. The former was predominant, leading to endosomal and lysosomal compartments, as well as recycling to the plasmalemma. The clathrin-independent processes led to a smooth vesicular compartment adjacent to the plasmalemma resembling caveolae, which did not transport ligand deeper within the cell. Potassium depletion largely eliminated clathrin-dependent endocytosis, while not interfering with agonist-stimulated receptor movement into subplasmalemmal smooth vesicle compartments. These cellular endocytic events can be related to the established cycle of CCK receptor phosphorylation and dephosphorylation, which we have previously described (Klueppelberg, U. G., L. K. Gates, F. S. Gorelick, and L. J. Miller. 1991. J. Biol. Chem. 266:2403-2408; Lutz, M. P., D. I. Pinon, L. K. Gates, S. Shenolikar, and L. J. Miller. 1993. J. Biol. Chem. 268:12136-12142). The rapid onset and peak of receptor phosphorylation after agonist occupation correlates best with a plasmalemmal localization, while stimulated receptor phosphatase activity correlates best with receptor residence in intracellular compartments. We postulate that the smooth vesicular compartment adjacent to the plasmalemma functions for the rapid resensitization of the receptor, while the classical clathrin-mediated endocytotic pathway is key for receptor downregulation via lysosomal degradation, as well as less rapid resensitization.

Animals↗

PubMed1995

Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell.

Receptor desensitization is a key process for the protection of the cell from continuous or repeated exposure to high concentrations of an agonist. Well-established mechanisms for desensitization of guanine nucleotide-binding protein (G protein)-coupled receptors include phosphorylation, sequestration/internalization, and down-regulation. In this work, we have examined some mechanisms for desensitization of the cholecystokinin (CCK) receptor which is native to the pancreatic acinar cell, and have found the predominant mechanism to be distinct from these recognized processes. Upon fluorescent agonist occupancy of the native receptor, it becomes "insulated" from the effects of acid washing and becomes immobilized on the surface of the plasma membrane in a time- and temperature-dependent manner. This localization was assessed by ultrastructural studies using a colloidal gold conjugate of CCK, and lateral mobility of the receptor was assessed using fluorescence recovery after photobleaching. Of note, recent application of the same morphologic techniques to a CCK receptor-bearing Chinese hamster ovary cell line demonstrated prominent internalization via the clathrin-dependent endocytic pathway, as well as entry into caveolae (Roettger, B.F., R.U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J.M. Larkin, and L.J. Miller, 1995, J. Cell Biol. 128: 1029-1041). These organelles are not observed to represent prominent compartments for the same receptor to traverse in the acinar cell, although fluorescent insulin is clearly internalized in these cells via receptor-mediated endocytosis. In this work, the rate of lateral mobility of the CCK receptor is observed to be similar in both cell types (1-3 x 10(-10) cm2/s), while the fate of the agonist-occupied receptor is quite distinct in each cell. This supports the unique nature of desensitization processes which occur in a cell-specific manner. A plasmalemmal site of insulation of this important receptor on the pancreatic acinar cell could be particularly effective to protect the cell from processes which might initiate pancreatitis, while providing for the rapid resensitization of this receptor to ensure appropriate pancreatic secretion to aid in nutrient assimilation for the organism.

Acids↗

PubMed1989

Hydrophobic interaction chromatography.

Hydrophobic interaction chromatography (HIC) is emerging as a useful technique for the separation of biological compounds. Advances in the past two years in HIC applications, stationary phases, eluents, and theory are reviewed. Recent applications of HIC processes include analytical and semi-preparative separations of a variety of proteins, such as isolectins, hemoglobins, calmodulin, and cardiotoxins. Additionally, HIC is being employed as a tool to investigate protein properties and mechanisms. Advances in HIC stationary phases include development of non-porous, microparticulate supports as well as supports with pore sizes up to 1000 Angstroms. Studies of HIC eluents have further shown the effects of mobile phase pH, water-structuring characterization, and surface tension increments on retention. Various retention mechanisms which have been presented are reviewed; and a correlation relating resolution to column and solute parameters is presented. Protein conformational effects at specific sites have been shown to have a significant impact on retention and specific examples illustrating such effects are discussed.

Journal Article↗