The circumstances in which recurrent laryngeal nerve palsy occurs after surgery for benign thyroid disease: a retrospective study of 1026 patients.

OBJECTIVE: To evaluate the circumstances in which recurrent laryngeal nerve palsy occurs after thyroid surgery. METHODS: This study assessed 1026 patients who underwent surgery for benign thyroid disease over a seven-year period in a retrospective, single-centre study. RESULTS: With a total of 1835 recurrent laryngeal nerves at risk, there were 38 cases (2.07 per cent) of transient recurrent laryngeal nerve palsy and 8 (0.44 per cent) of permanent recurrent laryngeal nerve palsy. No explanation was found for 10 of the 46 cases of recurrent laryngeal nerve palsy. Among the 38 other cases, the probable causes included poor identification of the recurrent laryngeal nerve during surgery, involuntary resection of the nerve and several other factors. CONCLUSION: Apart from accidental resection of the recurrent laryngeal nerve during thyroid surgery, the causes of post-operative recurrent laryngeal nerve palsy are often unclear and likely multifactorial. Poor identification of the recurrent laryngeal nerve during surgery is still the main cause of post-operative recurrent laryngeal nerve palsy, even when intra-operative neuromonitoring is used.

Identification of peptide ligand-binding domains within the human motilin receptor using photoaffinity labeling.

The cDNA encoding the human motilin receptor was recently cloned and found to represent a G protein-coupled receptor that is structurally related to the growth hormone secretagogue receptors. Together, these represent a new Class I receptor family. Our aim in the present work is to gain insight into the molecular basis of binding of motilin to its receptor using photoaffinity labeling. To achieve this, we developed a Chinese hamster ovary cell line that overexpressed functional motilin receptor (CHO-MtlR; 175,000 sites per cell, with K(i) = 2.3 +/- 0.4 nm motilin and EC(50) = 0.3 +/- 0.1 nm motilin) and a radioiodinatable peptide analogue of human motilin that incorporated a photolabile p-benzoyl-l-phenylalanine (Bpa) residue into its pharmacophoric domain. This probe, [Bpa(1),Ile(13)]motilin, was a full agonist at the motilin receptor that increased intracellular calcium in a concentration-dependent manner (EC(50) = 1.5 +/- 0.4 nm). This photolabile ligand bound specifically and with high affinity to the motilin receptor (K(i) = 12.4 +/- 1.0 nm), and covalently labeled that molecule within its M(r) = 45,000 deglycosylated core. Cyanogen bromide cleavage demonstrated its covalent attachment to fragments of the receptor having apparent M(r) = 6,000 and M(r) = 31,000. These were demonstrated to represent fragments that included both the first and the large second extracellular loop domains, with the latter representing a unique structural feature of this receptor. The spatial approximation of the pharmacophoric domain of motilin with these receptor domains support their functional importance as well.

Refinement of the structure of the ligand-occupied cholecystokinin receptor using a photolabile amino-terminal probe.

Affinity labeling is a powerful tool to establish spatial approximations between photolabile residues within a ligand and its receptor. Here, we have utilized a cholecystokinin (CCK) analogue with a photolabile benzoylphenylalanine (Bpa) sited in position 24, adjacent to the pharmacophoric domain of this hormone (positions 27-33). This probe was a fully efficacious agonist that bound to the CCK receptor saturably and with high affinity (K(i) = 8.9 +/- 1.1 nm). It covalently labeled the CCK receptor either within the amino terminus (between Asn(10) and Lys(37)) or within the third extracellular loop (Glu(345)), as demonstrated by proteolytic peptide mapping, deglycosylation, micropurification, and Edman degradation sequencing. Truncation of the receptor to eliminate residues 1-30 had no detrimental effect on CCK binding, stimulated signaling, or affinity labeling through a residue within the pharmacophore (Bpa(29)) but resulted in elimination of the covalent attachment of the Bpa(24) probe to the receptor. Thus, the distal amino terminus of the CCK receptor resides above the docked ligand, compressing the portion of the peptide extending beyond its pharmacophore toward the receptor core. Exposure of wild type and truncated receptor constructs to extracellular trypsin damaged the truncated construct but not the wild type receptor, suggesting that this domain also may play a protective role. Use of these additional insights into molecular approximations provided key constraints for molecular modeling of the peptide-receptor complex, supporting the counterclockwise organization of the transmembrane helical domains.

Cross-chimeric analysis of selectivity of secretin and VPAC(1) receptor activation.

Agonist action at receptors is highly specific, affected by the structure of both ligand and receptor. Chimeric constructs of structurally related receptors and/or ligands that have biological differences provide an opportunity to correlate a specific structural domain with function. In this work, we have used a cross-chimeric approach to explore the structural basis for rat secretin and vasoactive intestinal polypeptide action at their closely related secretin and VPAC(1) receptors, belonging to class II of the G protein-coupled receptor superfamily. Multiple domains of both ligands and receptors contributed toward their selectivity, with differing combinations of such domains able to support high-potency interactions. The amino-terminal 15 residues of secretin were most critical for potent stimulation of secretin receptors, whereas either the amino- or carboxyl-terminal halves of vasoactive intestinal polypeptide, when complemented by Lys(15), provided potent stimulation of the VPAC(1) receptor. The amino terminus of the VPAC(1) receptor was most critical for potent response to vasoactive intestinal polypeptide, whereas the amino terminus of the secretin receptor was important, but not adequate, requiring the complementation of an extracellular loop domain for potent response to secretin. Differences in the distribution of these determinants within these receptors provided an opportunity to produce a more "universal" receptor that contained the first extracellular loop of the secretin receptor and the remainder of the VPAC(1) receptor. This cross-chimeric approach should be applied to other members of this receptor family to test the emerging themes and to expand these insights as broadly as possible.

Identification of two pairs of spatially approximated residues within the carboxyl terminus of secretin and its receptor.

The carboxyl-terminal domains of secretin family peptides have been shown to contain key determinants for high affinity binding to their receptors. In this work, we have examined the interaction between carboxyl-terminal residues within secretin and the prototypic secretin receptor. We previously utilized photoaffinity labeling to demonstrate spatial approximation between secretin residue 22 and the receptor domain that includes the first 30 residues of the amino terminus (Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903-909). Here, we further refined the site of labeling with the p-benzoyl-phenylalanine (Bpa(22)) probe to receptor residue Leu(17) using progressive cleavage of wild type and mutant secretin receptors (V13M and V16M) and sequence analysis. We also developed a new probe incorporating a photolabile Bpa at position 26 of secretin, closer to its carboxyl terminus. This analogue was also a potent agonist (EC(50) = 72 +/- 6 pm) and bound to the secretin receptor specifically and with high affinity (K(i) = 10.3 +/- 2.4 nm). It covalently labeled the secretin receptor at a single site saturably and specifically. This was localized to the segment between residues Gly(34) and Ala(41) using chemical and enzymatic cleavage of labeled wild type and A41M mutant receptor constructs and immunoprecipitation of epitope-tagged receptor fragments. Radiochemical sequencing identified the site of covalent attachment as residue Leu(36). These new insights, along with our recent report of contact between residue 6 within the amino-terminal half of secretin and this same amino-terminal region of this receptor (Dong, M., Wang, Y., Hadac, E. M., Pinon, D. I., Holicky, E. L., and Miller, L. J. (1999) J. Biol. Chem. 274, 19161-19167), support a key role for this region, making the molecular details of this interaction of major interest.

Identification of an interaction between residue 6 of the natural peptide ligand and a distinct residue within the amino-terminal tail of the secretin receptor.

Photoaffinity labeling is a powerful tool for the characterization of the molecular basis of ligand binding. We recently used this technique to demonstrate the proximity between a residue within the carboxyl-terminal half of a secretin-like ligand and the amino-terminal domain of the secretin receptor (Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903-909). In this work, we have developed another novel radioiodinatable secretin analogue ([Bpa6,Tyr10]rat secretin-27) that incorporates a photolabile p-benzoyl-L-phenylalanine (Bpa) residue into position 6 of the amino-terminal half of the ligand and used this to identify a specific receptor residue proximate to it. This probe specifically bound to the secretin receptor with high affinity (IC50 = 13.2 +/- 2.5 nM) and was a potent stimulant of cAMP accumulation in secretin receptor-bearing Chinese hamster ovary-SecR cells (EC50 = 720 +/- 230 pM). It covalently labeled the secretin receptor in a saturable and specific manner. Cyanogen bromide cleavage of this molecule yielded a single labeled fragment that migrated on an SDS-polyacrylamide gel at Mr = 19,000 that shifted to 10 after deglycosylation, most consistent with either of two glycosylated fragments within the amino-terminal tail. By immunoprecipitation with antibody directed to epitope tags incorporated into each of the two candidate fragments, the most distal fragment at the amino terminus was identified as the domain of labeling. The labeled domain was further refined to the first 16 residues by endoproteinase Lys-C cleavage and by cyanogen bromide cleavage of another receptor construct in which Val16 was mutated to Met. Radiochemical sequencing of photoaffinity-labeled secretin receptor fragments established that Val4 was the specific site of covalent attachment. This provides the first residue-residue contact between a secretin ligand and its receptor and will contribute substantially to the molecular understanding of this interaction.

A peptide agonist acts by occupation of a monomeric G protein-coupled receptor: dual sites of covalent attachment to domains near TM1 and TM7 of the same molecule make biologically significant domain-swapped dimerization unlikely.

Membrane receptor dimerization is a well-established event for initiation of signaling at growth factor receptors and has been postulated to exist for G protein-coupled receptors, based on correction of nonfunctional truncated, mutant, or chimeric constructs by coexpression of appropriate normal complementary receptor domains. In this work, we have directly explored the molecular composition of the minimal functional unit of an agonist ligand and the wild-type G protein-coupled cholecystokinin (CCK) receptor, using photoaffinity labeling with a CCK analogue probe incorporating dual photolabile benzoylphenylalanine (Bpa) residues as sites of covalent attachment. This probe, 125I-D-Tyr-Gly-[(Nle28, 31, Bpa29,33)CCK-26-33], was shown to represent a full agonist and to specifically label the CCK receptor. Like probes incorporating individual photolabile residues in these positions,1,2 the two Bpa residues in the dual photoprobe covalently labeled receptor domains in the amino-terminal tail outside TM1 and in the third extracellular loop outside TM7. Absence of demonstrable receptor dimerization after the establishment of dual sites of covalent attachment supports the presence of these two domains within a single receptor molecule. Demonstration of the covalent adduct of a single probe molecule with the two cyanogen bromide fragments of the CCK receptor representing the expected domains further supports this interpretation. Thus, while domain-swapped dimerization of G protein-coupled receptors may be possible as a mechanism of rescue for nonfunctional molecules, it is not necessary for ligand binding and initiation of signaling at a wild-type receptor in this superfamily. The functional unit for CCK action is normally a ligand-receptor monomer.

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.

Structurally related peptide agonist, partial agonist, and antagonist occupy a similar binding pocket within the cholecystokinin receptor. Rapid analysis using fluorescent photoaffinity labeling probes and capillary electrophoresis.

The molecular basis of ligand binding to receptors provides important insights for drug development. Here, we explore domains of the cholecystokinin (CCK) receptor that are critical for ligand binding, using a novel series of fluorescent photolabile probes, receptor proteolysis, and rapid high resolution separation of peptide fragments by capillary electrophoresis. Each probe incorporated the same fluorophore and a photolabile p-benzoylphenylalanine at the amino terminus of the pharmacophoric domain (residue 24 of CCK-33) of CCK analogues representing full agonist, partial agonist, and antagonist of this receptor. Each was used to label the CCK receptor expressed on Chinese hamster ovary-CCKR cells, with the labeled domain then released by cyanogen bromide cleavage. Capillary electrophoresis with laser-induced fluorescence detection achieved an on-capillary mass sensitivity of 1.6 attomoles (10(-18) mol), with an excellent signal-to-noise ratio. Each of the biologically divergent, but structurally similar probes saturably and specifically labeled the same receptor domain, consistent with conservation of "docking" determinants. This had an apparent mass of 2.9 kDa, most consistent with the first extracellular loop domain. An additional probe having its site of covalent attachment in a different region of the probe (residue 29 of CCK-33) labeled a distinct receptor fragment with differential migration on capillary electrophoresis (third extracellular loop). Identification of the specific receptor residue(s) covalently linked to the amino-terminal probes must await further fragmentation and sequence analysis.

Demonstration of a direct interaction between residue 22 in the carboxyl-terminal half of secretin and the amino-terminal tail of the secretin receptor using photoaffinity labeling.

An understanding of the molecular basis of hormonal activation of receptors provides important insights for drug design. Toward this end, intrinsic photoaffinity labeling is a powerful tool to directly identify the ligand-binding domain. We have developed a new radioiodinatable agonist ligand of the secretin receptor that incorporates a photolabile p-benzoyl-L-phenylalanine (Bpa) into the position of Leu22 and have utilized this to identify the adjacent receptor domain. The rat [Tyr10,Bpa22]secretin-27 probe was a fully efficacious agonist, with a potency to stimulate cAMP accumulation by Chinese hamster ovary SecR cells similar to that of natural secretin (EC50 = 68 +/- 22 pM analogue and 95 +/- 25 pM secretin). It bound specifically and with high affinity (Ki = 5.0 +/- 1.1 nM) and covalently labeled the Mr = 57,000-62,000 secretin receptor. Cyanogen bromide cleavage of the receptor yielded a major labeled fragment of apparent Mr = 19,000 that shifted to Mr = 9,000 after deglycosylation. This was most consistent with either of two glycosylated domains within the amino-terminal tail of the receptor. Immunoprecipitation with antibody directed to epitope tags incorporated into each of the candidate domains established that the fragment at the amino terminus of the receptor was the site of labeling. This was further localized to the amino-terminal 30 residues of the receptor by additional proteolysis of this fragment with endoproteinase Lys-C. This provides the first direct demonstration of a contact between a secretin-like agonist and its receptor and will contribute a useful constraint to the modeling of this interaction.

Direct identification of a second distinct site of contact between cholecystokinin and its receptor.

We have developed a biologically active analogue of cholecystokinin (CCK) that incorporates a photolabile benzoylphenylalanine (Bpa) moiety in the middle of its pharmacophoric domain, which efficiently establishes a covalent bond with an interacting domain of the CCK receptor. This probe incorporated L-Bpa in the position of Gly29 of the well characterized, radioiodinatable CCK analogue, D-Tyr-Gly-[(Nle28,31)CCK-26-33]. It was a potent pancreatic secretagogue (EC50 = 28 +/- 6 nM) that was equally efficacious with natural CCK, and bound to the CCK receptor with moderate affinity (IC50 = 450 +/- 126 nM). This was adequate to allow specific covalent labeling of the receptor. The labeled domain was within the cyanogen bromide fragment of the receptor including the top of TM6 (the sixth transmembrane domain), the third extracellular loop, and TM7 (the seventh transmembrane domain), as proven by direct Edman degradation sequencing. When this fragment was modified by the replacement of Val342 with Met to generate an additional site of cyanogen bromide cleavage, the labeled fragment was reduced in apparent size consistent with its representing the carboxyl-terminal portion of this fragment. Radiochemical sequencing of that fragment demonstrated covalent attachment of the probe to His347 and Leu348 in this domain. This represents the second experimentally demonstrated contact between a CCK analogue and this receptor, complementing the labeling of the domain just above TM1 (the first transmembrane domain) by a photolabile residue at the carboxyl terminus of CCK (Ji, Z. S., Hadac, E. M., Henne, R. M., Patel, S. A., Lybrand, T. P., and Miller, L. J. (1997) J. Biol. Chem. 272, 24393-24401). Both contacts are consistent with the conformational model of CCK binding proposed on the basis of the initial contact.

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.

[Variability in esterases of Metarhizium anisopliae]. / Variabilidad de las esterasas de Metarhizium anisopliae.

The variability in esterases of the entomogenous fungus Metarhizium anisopliae was determined electrophoretically on 8.5% polyacrylamide gel. Ten isolates from diverse taxonomic groups of insects were analyzed. The electrophoretic analysis showed differences and similarities between these isolates and it was possible to distinguish six different patterns. The results obtained show a great polymorphism for the esterase system of M. anisopliae.

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.

Development of magnetic beads for rapid and efficient metal-chelate affinity purifications.

Metal-chelate affinity chromatography offers multiple advantages for protein purification, yet existing resins make its applications to sparse, hydrophobic, or particularly labile proteins and peptides quite difficult. In this work, we have developed a simple method to covalently modify commercially available superparamagnetic beads with a six-carbon spacer and nitrilotriacetic acid to provide a novel resin for extremely rapid and efficient metal chelate affinity purifications. Further, the small size and surface chemistry of these beads provide clear improvement in applicability to small scale purifications with reduced nonspecific adsorption. These advantages have been demonstrated relative to a commercially available nickel resin.

Relationship between native and recombinant cholecystokinin receptors: role of differential glycosylation.

In an attempt to establish the relationship between the protein encoded by the recently cloned type A cholecystokinin (CCK) receptor cDNA and the two distinct plasmalemmal proteins on the rat pancreatic acinar cell that were previously described as candidates to represent this receptor, we have established a Chinese hamster ovary (CHO) cell line stably expressing large amounts of this recombinant protein and have used biochemical methods to characterize it directly. Upon affinity labeling, this protein migrated faster on a sodium dodecyl sulfate-polyacrylamide gel than the M(r) 85,000-95,000 molecule previously felt to represent the best candidate. However, deglycosylation with endoglycosidase F demonstrated that it had the same size core protein as that candidate, and this identification was further supported by protease peptide mapping. We postulated that the structural differences between the recombinant and the native proteins related to differences in glycosylation. Consistent with this, lectin-binding experiments demonstrated that both represented complex glycoproteins but that only the native receptor-bound Ulex europeus agglutinin I. Since this lectin binds to fucose residues that are added late in glycoprotein biosynthesis, it is possible that the distinct processing observed affected only that step. In spite of this structural difference, the type A CCK receptor-bearing CHO cell CCK receptor was functionally indistinguishable from the native acinar cell receptor. This included its ability to initiate signaling cascades, its sensitivity to stable GTP analogues, and its binding affinities for agonists and antagonists. The fidelity of this receptor expression system, while representing a 25-fold increase in receptor density over the native pancreatic acinar cell, should provide an ideal substrate for the examination of structure-function relationships within this molecule.

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.

A nonradioactive fluorescent gel-shift assay for the analysis of protein phosphatase and kinase activities toward protein-specific peptide substrates.

Synthetic peptides are important tools with which to study the activities of protein kinases and phosphatases toward specific substrate sequences which are present within selected regions of a protein. Most existing assays for the phosphorylation or dephosphorylation of such peptides utilize 32P and either affinity chromatography or HPLC separation and require extensive characterization and validation. Here, we describe a method for monitoring the phosphorylation or dephosphorylation of almost any peptide of interest which does not require the use of radioactivity, making its reagents stable for a prolonged period, and which can be performed in any standard laboratory. For this, after performance of kinase or phosphatase reactions with the peptide of interest, products are derivatized with fluorescamine and are separated according to charge by agarose gel electrophoresis. Phosphorylated and nonphosphorylated peptides are readily separated and can be both identified and quantified by uv detection. The lower limit for detection of peptide in the agarose gel was 0.02 nmol using the gel-shift kinase assay with cAMP-dependent kinase and Kemptide as substrate. This had sensitivity and reproducibility similar to those of a standard assay using [gamma-32P]ATP with this substrate. Dephosphorylation of a synthetic phosphopeptide corresponding to a segment of the cholecystokinin receptor was tested in an analogous assay with known amounts of protein phosphatase 2A. Phosphopeptide and dephosphopeptide were easily detected and quantified with as little as 0.03 mU/ml protein phosphatase 2A activity. Therefore, with this assay, most synthetic peptides and phosphopeptides can be used as substrates without further modification. This will be of particular interest for monitoring the purification of highly specific protein kinase and phosphatase activities.

Characterization of protein serine/threonine phosphatases in rat pancreas and development of an endogenous substrate-specific phosphatase assay.

Protein phosphatases have recently been recognized to represent an important, independently regulated portion of cellular signaling cascades. Although reversible phosphorylation of multiple pancreatic proteins has been described, suggesting a role for these enzymes, little is known about the characteristics of protein phosphatases in this organ. In this work, we have characterized and quantified the serine/threonine phosphatases present in pancreatic cytosol and plasma membranes. Using a sensitive and specific in vitro assay with standard substrates (phosphorylase a and phosphocasein), the predominant enzymes represented protein phosphatase 2A in cytosol and protein phosphatase 1 in plasma membranes, with both compartments having substantial amounts of both of these enzymes. Both compartments also had protein phosphatase 2B activity, whereas protein phosphatase 2C was only measurable in the plasma membrane fraction. Further, a novel assay was developed and validated in which the action of an endogenous protein phosphatase on a specific cellular phosphoprotein was studied. For this, we utilized as substrate the cholecystokinin receptor which had been phosphorylated in response to agonist stimulation of the intact acinar cell. This type of assay will be key for the analysis of the mediation and regulation of dephosphorylation events which actually occur in the cell.

Intrinsic photoaffinity labeling of native and recombinant rat pancreatic secretin receptors.

BACKGROUND: Structural characterization of pancreatic secretin receptors has been limited by difficulties in generating suitable radioligands and obtaining sufficient substrate. The aims of this study were to design, synthesize, and characterize high affinity radiolabeled analogues of secretin suitable for "intrinsic" photoaffinity labeling and to clone, express, and characterize the recombinant rat pancreatic secretin receptor. METHODS: The ability of synthetic analogues to stimulate amylase secretion by pancreatic acini was studied. Receptor complementary DNA (cDNA) was cloned by screening a rat pancreatic library with a probe based on the sequence of a neural cell secretin-binding protein. Competition binding and affinity labeling were performed with membranes prepared from rat pancreas and transfected cells. RESULTS: Two probes were fully efficacious secretagogues, which bound in a specific, high-affinity, rapid, and temperature-dependent manner. Only ([125I]Tyr10, pNO2-Phe22) rat secretin 27 covalently labeled a 50,000-62,000-molecular weight pancreatic membrane protein, with labeling inhibited in a concentration-dependent manner by secretin but not vasoactive intestinal polypeptide. Hybridization screening yielded a full-length cDNA identical to the neural clone. Photoaffinity labeling of this recombinant receptor identified a 57,000-62,000-molecular weight protein with specificity similar to that of native pancreas. Both native and recombinant receptors migrated at a molecular weight of 42,000 after endoglycosidase F deglycosylation. CONCLUSIONS: This study provides evidence for the molecular identity of the pancreatic secretin receptor and presents a novel probe important in structural characterization of its agonist-binding domain.