Gerry Baquiran - Academia.edu (original) (raw)
Papers by Gerry Baquiran
Previous work has shown that bpV (phen) induces potent insulin-mimicking effects in the rat; sele... more Previous work has shown that bpV (phen) induces potent insulin-mimicking effects in the rat; selectively activates the endosomal (EN) insulin receptor kinase (IRK) in liver; and markedly abolishes endosomal IRK-associated PTP activity (IRK-aPTP) while reducing that of total ENs by ~ 30%. Here we examined the relatively selective effect of bpV (phen) on endosomal PTP activities for the purpose of defining IRK-aPTP(s). Using an in-gel PTP assay we detected multiple (~20) species of endosomal PTP (30 to > 220 kDa) with 5 being markedly inhibited following in vivo bpV(phen) administration. Using a combination of Mono Q anionic exchange chromatography and immunoblotting we demonstrated that LAR, PTP-α, and PTP-1B were present in endosomal subfractions not significantly inhibited by bpV (phen). PTP-1B activity was assayed in immunoprecipitates from hepatic ENs of control and bpV(phen)-treated rats and was found to be inhibited by ~30 % following bpV (phen) treatment. To clarify further the
Endocrinology, Dec 1, 2000
Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling protei... more Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling proteins between subcellular compartments in rat liver. In plasma membranes (PM) and microsomes, insulin induced a rapid decrease in insulin receptor substrate-1/2 (IRS1/2) within 30 sec and an increase in these proteins in endosomes (EN) and cytosol. The level of p85 in PM increased 2.3-fold at 30 sec after insulin stimulation followed by a decrease at 2 min. In this interval, 60 -85% and 10 -20% of p85 in PM was associated with IRS1 and IRS2, respectively. Thus, in PM, IRS1/2 accounts for almost all of the protein involved in phosphatidylinositol 3-kinase activation. In ENs insulin induced a maximal increase of 40% in p85 recruitment. As in PM, almost all p85 was associated with IRS1/2. The greater level of p85 recruitment to PM was associated with a higher level of insulininduced recruitment of Akt1 to this compartment (4.0-fold in PM vs. 2.4-fold in EN). There was a close correlation between Akt1 activity and Akt1 phosphorylation at Thr 308 and Ser 473 in PM and cytosol. However, in ENs the level of Akt1 activity per unit of phosphorylated Akt1 was significantly greater than in PM, indicating that in addition to phosphorylation, another factor(s) modulates Akt1 activation by insulin in rat liver. Our results demonstrate that activation of the insulin receptor kinase and modulation of key components of the insulin signaling cascade occur at the cell surface and within the endosomal system. These data provide further support for the role of the endocytic process in cell signaling. (Endocrinology 141: [4041][4042][4043][4044][4045][4046][4047][4048][4049] 2000)
In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary... more In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary and sufficient to account for epidermal growth factor (EGF)-induced DNA synthesis. In these cells, three major p85-containing complexes were formed after EGF treatment: ErbB3-p85, Shc-p85, and a multimeric Gab2-Grb2-SHP2-p85, which accounted for more than 80% of total EGF-induced PI3K activity (Kong, M., C. Mounier, J. Wu, and B. I. Posner, J Biol Chem, 2000, 275:36035-36042). More recently, we found that EGF-dependent tyrosine phosphorylation of endogenous Gab2 is essential for EGF-induced DNA synthesis in rat hepatocytes. Here we show that, after EGF treatment, ErbB3-p85 and Shc-p85 complexes were localized to plasma membrane and endosomes, whereas the multimeric Gab2-Grb2-SHP2-p85 complex was formed rapidly (peak at 30 sec) and exclusively in cytosol. Western blotting of subcellular fractions from intact liver and immunofluorescence analyses in cultured hepatocytes demonstrated that EGF did not promote the association of cytosolic Gab2 with cell membranes. These observations prompted us to evaluate the role of the PH domain of Gab2 in regulating its function. Overexpression of the PH domain of Gab2 did not affect EGF-induced Gab2 phosphorylation, PI3K activation, and DNA synthesis. Overexpressed Gab2 lacking the PH domain (⌬PHGab2) was comparable to wild-type Gab2 in respect to EGF-induced tyrosine phosphorylation, recruitment of p85, and DNA synthesis. In summary, after EGF stimulation, ErbB3, Shc, and Gab2 are differentially compartmentalized in rat liver, where they associate with and activate PI3K. Our data demonstrate that Gab2 mediates EGF-induced PI3K activation and DNA synthesis in a PH domain-independent manner. (Molecular Endocrinology 17: 935-944, 2003)
Previous work has shown that bpV (phen) induces potent insulin-mimicking effects in the rat; sele... more Previous work has shown that bpV (phen) induces potent insulin-mimicking effects in the rat; selectively activates the endosomal (EN) insulin receptor kinase (IRK) in liver; and markedly abolishes endosomal IRK-associated PTP activity (IRK-aPTP) while reducing that of total ENs by ~ 30%. Here we examined the relatively selective effect of bpV (phen) on endosomal PTP activities for the purpose of defining IRK-aPTP(s). Using an in-gel PTP assay we detected multiple (~20) species of endosomal PTP (30 to > 220 kDa) with 5 being markedly inhibited following in vivo bpV(phen) administration. Using a combination of Mono Q anionic exchange chromatography and immunoblotting we demonstrated that LAR, PTP-α, and PTP-1B were present in endosomal subfractions not significantly inhibited by bpV (phen). PTP-1B activity was assayed in immunoprecipitates from hepatic ENs of control and bpV(phen)-treated rats and was found to be inhibited by ~30 % following bpV (phen) treatment. To clarify further the role of PTP-1B in dephosphorylating IRK we prepared hepatic ENs from wild type and PTP-1B null mice and found that the phosphotyrosine content of IRK was similar in these two types of ENs, and that IRK dephosphorylation was not affected in ENs from PTP-1B null mice compared to that in ENs from wild type mice. These data suggest that LAR, PTP-α, and PTP-1B are not candidates for the IRK-associated PTP in hepatic ENs, and that IRK dephosphorylation in ENs may result from the concerted action of several PTPs.
Proceedings of the National Academy of Sciences, 1985
Adrenocorticotropin binding sites in the rat median eminence have been localized in vivo. These b... more Adrenocorticotropin binding sites in the rat median eminence have been localized in vivo. These binding sites occur in the basalar zone, which is rich in axonal endings. Using competitive binding and quantitative light-microscope radioautography, we found that the median-eminence binding site, in contradistinction to the adrenal receptor, binds specifically the residue 4-10 region of the adrenocorticotropin molecule. Using quantitative electron-microscope radioautography and median-eminence deafferentation, we localized the binding sites to axon terminals in this region. In time-delayed uptake studies using light-microscope radioautography, we failed to observe concentration of radiolabel in neurons of the medial basal hypothalamus after the direct injection of radioiodinated adrenocorticotropin(1-24) into the median eminence.
Gab2 Tyrosine Phosphorylation by a Pleckstrin Homology Domain-Independent Mechanism: Role in Epidermal Growth Factor-Induced Mitogenesis
Molecular Endocrinology, 2003
In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary... more In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary and sufficient to account for epidermal growth factor (EGF)-induced DNA synthesis. In these cells, three major p85-containing complexes were formed after EGF treatment: ErbB3-p85, Shc-p85, and a multimeric Gab2-Grb2-SHP2-p85, which accounted for more than 80% of total EGF-induced PI3K activity (Kong, M., C. Mounier, J. Wu, and B. I. Posner, J Biol Chem, 2000, 275:36035-36042). More recently, we found that EGF-dependent tyrosine phosphorylation of endogenous Gab2 is essential for EGF-induced DNA synthesis in rat hepatocytes. Here we show that, after EGF treatment, ErbB3-p85 and Shc-p85 complexes were localized to plasma membrane and endosomes, whereas the multimeric Gab2-Grb2-SHP2-p85 complex was formed rapidly (peak at 30 sec) and exclusively in cytosol. Western blotting of subcellular fractions from intact liver and immunofluorescence analyses in cultured hepatocytes demonstrated that EGF did not promote the association of cytosolic Gab2 with cell membranes. These observations prompted us to evaluate the role of the PH domain of Gab2 in regulating its function. Overexpression of the PH domain of Gab2 did not affect EGF-induced Gab2 phosphorylation, PI3K activation, and DNA synthesis. Overexpressed Gab2 lacking the PH domain (DeltaPHGab2) was comparable to wild-type Gab2 in respect to EGF-induced tyrosine phosphorylation, recruitment of p85, and DNA synthesis. In summary, after EGF stimulation, ErbB3, Shc, and Gab2 are differentially compartmentalized in rat liver, where they associate with and activate PI3K. Our data demonstrate that Gab2 mediates EGF-induced PI3K activation and DNA synthesis in a PH domain-independent manner.
Endocrinology, 2000
Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling protei... more Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling proteins between subcellular compartments in rat liver. In plasma membranes (PM) and microsomes, insulin induced a rapid decrease in insulin receptor substrate-1/2 (IRS1/2) within 30 sec and an increase in these proteins in endosomes (EN) and cytosol. The level of p85 in PM increased 2.3-fold at 30 sec after insulin stimulation followed by a decrease at 2 min. In this interval, 60 -85% and 10 -20% of p85 in PM was associated with IRS1 and IRS2, respectively. Thus, in PM, IRS1/2 accounts for almost all of the protein involved in phosphatidylinositol 3-kinase activation. In ENs insulin induced a maximal increase of 40% in p85 recruitment. As in PM, almost all p85 was associated with IRS1/2. The greater level of p85 recruitment to PM was associated with a higher level of insulininduced recruitment of Akt1 to this compartment (4.0-fold in PM vs. 2.4-fold in EN). There was a close correlation between Akt1 activity and Akt1 phosphorylation at Thr 308 and Ser 473 in PM and cytosol. However, in ENs the level of Akt1 activity per unit of phosphorylated Akt1 was significantly greater than in PM, indicating that in addition to phosphorylation, another factor(s) modulates Akt1 activation by insulin in rat liver. Our results demonstrate that activation of the insulin receptor kinase and modulation of key components of the insulin signaling cascade occur at the cell surface and within the endosomal system. These data provide further support for the role of the endocytic process in cell signaling. (Endocrinology 141: [4041][4042][4043][4044][4045][4046][4047][4048][4049] 2000)
Endocrinology, 2006
lin-mimicking effects in the rat, selectively activates the endosomal (EN) insulin receptor kinas... more lin-mimicking effects in the rat, selectively activates the endosomal (EN) insulin receptor kinase (IRK) in liver, and markedly abolishes endosomal IRK-associated phosphotyrosine phosphatase (PTP) activity while reducing that of total ENs by approximately 30%. In this study we examined the relatively selective effect of bpv(phen) on endosomal PTP activities for the purpose of defining IRK-associated PTP(s). Using an in-gel PTP assay, we detected multiple (ϳ20) species of endosomal PTP (30 to >220 kDa), with five that were markedly inhibited after in vivo bpV(phen) administration. Using a combination of Mono Q anionic exchange chromatography and immunoblotting, we demonstrated that LAR (leukocyte common antigen-related), PTP-␣, and PTP-1B were present in endosomal subfractions not significantly inhibited by bpv-(phen). PTP-1B activity was assayed in immunoprecipitates from hepatic ENs of control and bpV(phen)-treated rats and was found to be inhibited by approximately 30% after bpv-(phen) treatment. To clarify the role of PTP-1B in dephosphorylating IRK, we prepared hepatic ENs from wild-type and PTP-1B-null mice. We found that the phosphotyrosine content of IRK was similar in these two types of ENs, and that IRK dephosphorylation was not affected in ENs from PTP-1B-null mice compared with that in ENs from wild-type mice. These data suggest that LAR , PTP-␣, and PTP-1B are not candidates for the IRK-associated PTP in hepatic ENs, and that IRK dephosphorylation in ENs may result from the concerted actions of several PTPs.
Quantitative in Vivo Autoradiographic Localization of [ 125 I_Tyr 11 ] Somatostatin-14-and [Leu 8 , d -Trp 22 - 125 ITyr- 125 ] Somatostatin-28-Binding Sites in Rat Brain*
Endocrinology, 1986
Quantitative in vivo autoradiography was used to identify and compare the regional distribution o... more Quantitative in vivo autoradiography was used to identify and compare the regional distribution of specific binding sites for blood-borne [125I-Tyr11]somatostatin-14 (S-14 section) and [Leu8,D-Trp22-125I-Tyr25]somatostatin-28 (S-28 section) in the rat brain. Rats were given intracardiac injections of 17 pmol S-14 section (with or without unlabeled S-14) or 17 pmol S-28 section (with or without unlabeled S-28). After whole body perfusion and fixation, brains were processed for light microscopic autoradiography of S-14 section- and S-28 section-binding sites. Of the peripheral tissues, the adrenal glands showed the highest uptake of S-14 section and S-28 section (determined by counting) and were subsequently processed for autoradiography for comparison with brain. Specific autoradiographic grains (ARG) associated with both radioligands were identified only in the circumventricular organs (CVOs). The highest ARG density associated with S-14 section was found in the area postrema, followed in decreasing order by the subfornical organ and the organum vasculosum lamina terminalis region. Median eminence (ME) contained virtually no specific S-14 section ARG. As with S-14 section, the highest ARG density of S-28 section-binding sites was also found in the area postrema, which labeled approximately equally with the two radioligands. This was followed by the ME, subfornical organ, and organum vasculosum lamina terminalis. The overall patterns of labeling of the CVOs with S-14 section and S-28 section showed significant differences, especially in the ME. Within the ME, labeled S-28 section was concentrated in a broad band throughout the external zone in a location identical to that of immunoreactive S-14. Analysis of dose-response curves obtained with 0.3-30 nmol unlabeled S-14 or S-28 revealed IC50 values for S-14 3- to 6-fold lower than those for S-28 for all labeled CVOs. With both S-14 section and S-28 section, the labelling density of the adrenal glands was double that of the area postrema. Adrenal binding of the radioligands was confined to the cells of the zona glomerulosa. We conclude: specific high affinity binding sites for S-14 section and S-28 section exist in the CVOs and the adrenal glomerulosa; and the 3- to 6-fold higher affinity of binding of S-14 to CVOs compared to S-28 together with the dissimilar patterns of labelling of the different CVOs by the two radioligands suggest the existence of separate populations of S-14 and S-28 receptors.
Previous work has shown that bpV (phen) induces potent insulin-mimicking effects in the rat; sele... more Previous work has shown that bpV (phen) induces potent insulin-mimicking effects in the rat; selectively activates the endosomal (EN) insulin receptor kinase (IRK) in liver; and markedly abolishes endosomal IRK-associated PTP activity (IRK-aPTP) while reducing that of total ENs by ~ 30%. Here we examined the relatively selective effect of bpV (phen) on endosomal PTP activities for the purpose of defining IRK-aPTP(s). Using an in-gel PTP assay we detected multiple (~20) species of endosomal PTP (30 to > 220 kDa) with 5 being markedly inhibited following in vivo bpV(phen) administration. Using a combination of Mono Q anionic exchange chromatography and immunoblotting we demonstrated that LAR, PTP-α, and PTP-1B were present in endosomal subfractions not significantly inhibited by bpV (phen). PTP-1B activity was assayed in immunoprecipitates from hepatic ENs of control and bpV(phen)-treated rats and was found to be inhibited by ~30 % following bpV (phen) treatment. To clarify further the
Endocrinology, Dec 1, 2000
Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling protei... more Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling proteins between subcellular compartments in rat liver. In plasma membranes (PM) and microsomes, insulin induced a rapid decrease in insulin receptor substrate-1/2 (IRS1/2) within 30 sec and an increase in these proteins in endosomes (EN) and cytosol. The level of p85 in PM increased 2.3-fold at 30 sec after insulin stimulation followed by a decrease at 2 min. In this interval, 60 -85% and 10 -20% of p85 in PM was associated with IRS1 and IRS2, respectively. Thus, in PM, IRS1/2 accounts for almost all of the protein involved in phosphatidylinositol 3-kinase activation. In ENs insulin induced a maximal increase of 40% in p85 recruitment. As in PM, almost all p85 was associated with IRS1/2. The greater level of p85 recruitment to PM was associated with a higher level of insulininduced recruitment of Akt1 to this compartment (4.0-fold in PM vs. 2.4-fold in EN). There was a close correlation between Akt1 activity and Akt1 phosphorylation at Thr 308 and Ser 473 in PM and cytosol. However, in ENs the level of Akt1 activity per unit of phosphorylated Akt1 was significantly greater than in PM, indicating that in addition to phosphorylation, another factor(s) modulates Akt1 activation by insulin in rat liver. Our results demonstrate that activation of the insulin receptor kinase and modulation of key components of the insulin signaling cascade occur at the cell surface and within the endosomal system. These data provide further support for the role of the endocytic process in cell signaling. (Endocrinology 141: [4041][4042][4043][4044][4045][4046][4047][4048][4049] 2000)
In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary... more In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary and sufficient to account for epidermal growth factor (EGF)-induced DNA synthesis. In these cells, three major p85-containing complexes were formed after EGF treatment: ErbB3-p85, Shc-p85, and a multimeric Gab2-Grb2-SHP2-p85, which accounted for more than 80% of total EGF-induced PI3K activity (Kong, M., C. Mounier, J. Wu, and B. I. Posner, J Biol Chem, 2000, 275:36035-36042). More recently, we found that EGF-dependent tyrosine phosphorylation of endogenous Gab2 is essential for EGF-induced DNA synthesis in rat hepatocytes. Here we show that, after EGF treatment, ErbB3-p85 and Shc-p85 complexes were localized to plasma membrane and endosomes, whereas the multimeric Gab2-Grb2-SHP2-p85 complex was formed rapidly (peak at 30 sec) and exclusively in cytosol. Western blotting of subcellular fractions from intact liver and immunofluorescence analyses in cultured hepatocytes demonstrated that EGF did not promote the association of cytosolic Gab2 with cell membranes. These observations prompted us to evaluate the role of the PH domain of Gab2 in regulating its function. Overexpression of the PH domain of Gab2 did not affect EGF-induced Gab2 phosphorylation, PI3K activation, and DNA synthesis. Overexpressed Gab2 lacking the PH domain (⌬PHGab2) was comparable to wild-type Gab2 in respect to EGF-induced tyrosine phosphorylation, recruitment of p85, and DNA synthesis. In summary, after EGF stimulation, ErbB3, Shc, and Gab2 are differentially compartmentalized in rat liver, where they associate with and activate PI3K. Our data demonstrate that Gab2 mediates EGF-induced PI3K activation and DNA synthesis in a PH domain-independent manner. (Molecular Endocrinology 17: 935-944, 2003)
Previous work has shown that bpV (phen) induces potent insulin-mimicking effects in the rat; sele... more Previous work has shown that bpV (phen) induces potent insulin-mimicking effects in the rat; selectively activates the endosomal (EN) insulin receptor kinase (IRK) in liver; and markedly abolishes endosomal IRK-associated PTP activity (IRK-aPTP) while reducing that of total ENs by ~ 30%. Here we examined the relatively selective effect of bpV (phen) on endosomal PTP activities for the purpose of defining IRK-aPTP(s). Using an in-gel PTP assay we detected multiple (~20) species of endosomal PTP (30 to > 220 kDa) with 5 being markedly inhibited following in vivo bpV(phen) administration. Using a combination of Mono Q anionic exchange chromatography and immunoblotting we demonstrated that LAR, PTP-α, and PTP-1B were present in endosomal subfractions not significantly inhibited by bpV (phen). PTP-1B activity was assayed in immunoprecipitates from hepatic ENs of control and bpV(phen)-treated rats and was found to be inhibited by ~30 % following bpV (phen) treatment. To clarify further the role of PTP-1B in dephosphorylating IRK we prepared hepatic ENs from wild type and PTP-1B null mice and found that the phosphotyrosine content of IRK was similar in these two types of ENs, and that IRK dephosphorylation was not affected in ENs from PTP-1B null mice compared to that in ENs from wild type mice. These data suggest that LAR, PTP-α, and PTP-1B are not candidates for the IRK-associated PTP in hepatic ENs, and that IRK dephosphorylation in ENs may result from the concerted action of several PTPs.
Proceedings of the National Academy of Sciences, 1985
Adrenocorticotropin binding sites in the rat median eminence have been localized in vivo. These b... more Adrenocorticotropin binding sites in the rat median eminence have been localized in vivo. These binding sites occur in the basalar zone, which is rich in axonal endings. Using competitive binding and quantitative light-microscope radioautography, we found that the median-eminence binding site, in contradistinction to the adrenal receptor, binds specifically the residue 4-10 region of the adrenocorticotropin molecule. Using quantitative electron-microscope radioautography and median-eminence deafferentation, we localized the binding sites to axon terminals in this region. In time-delayed uptake studies using light-microscope radioautography, we failed to observe concentration of radiolabel in neurons of the medial basal hypothalamus after the direct injection of radioiodinated adrenocorticotropin(1-24) into the median eminence.
Gab2 Tyrosine Phosphorylation by a Pleckstrin Homology Domain-Independent Mechanism: Role in Epidermal Growth Factor-Induced Mitogenesis
Molecular Endocrinology, 2003
In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary... more In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary and sufficient to account for epidermal growth factor (EGF)-induced DNA synthesis. In these cells, three major p85-containing complexes were formed after EGF treatment: ErbB3-p85, Shc-p85, and a multimeric Gab2-Grb2-SHP2-p85, which accounted for more than 80% of total EGF-induced PI3K activity (Kong, M., C. Mounier, J. Wu, and B. I. Posner, J Biol Chem, 2000, 275:36035-36042). More recently, we found that EGF-dependent tyrosine phosphorylation of endogenous Gab2 is essential for EGF-induced DNA synthesis in rat hepatocytes. Here we show that, after EGF treatment, ErbB3-p85 and Shc-p85 complexes were localized to plasma membrane and endosomes, whereas the multimeric Gab2-Grb2-SHP2-p85 complex was formed rapidly (peak at 30 sec) and exclusively in cytosol. Western blotting of subcellular fractions from intact liver and immunofluorescence analyses in cultured hepatocytes demonstrated that EGF did not promote the association of cytosolic Gab2 with cell membranes. These observations prompted us to evaluate the role of the PH domain of Gab2 in regulating its function. Overexpression of the PH domain of Gab2 did not affect EGF-induced Gab2 phosphorylation, PI3K activation, and DNA synthesis. Overexpressed Gab2 lacking the PH domain (DeltaPHGab2) was comparable to wild-type Gab2 in respect to EGF-induced tyrosine phosphorylation, recruitment of p85, and DNA synthesis. In summary, after EGF stimulation, ErbB3, Shc, and Gab2 are differentially compartmentalized in rat liver, where they associate with and activate PI3K. Our data demonstrate that Gab2 mediates EGF-induced PI3K activation and DNA synthesis in a PH domain-independent manner.
Endocrinology, 2000
Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling protei... more Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling proteins between subcellular compartments in rat liver. In plasma membranes (PM) and microsomes, insulin induced a rapid decrease in insulin receptor substrate-1/2 (IRS1/2) within 30 sec and an increase in these proteins in endosomes (EN) and cytosol. The level of p85 in PM increased 2.3-fold at 30 sec after insulin stimulation followed by a decrease at 2 min. In this interval, 60 -85% and 10 -20% of p85 in PM was associated with IRS1 and IRS2, respectively. Thus, in PM, IRS1/2 accounts for almost all of the protein involved in phosphatidylinositol 3-kinase activation. In ENs insulin induced a maximal increase of 40% in p85 recruitment. As in PM, almost all p85 was associated with IRS1/2. The greater level of p85 recruitment to PM was associated with a higher level of insulininduced recruitment of Akt1 to this compartment (4.0-fold in PM vs. 2.4-fold in EN). There was a close correlation between Akt1 activity and Akt1 phosphorylation at Thr 308 and Ser 473 in PM and cytosol. However, in ENs the level of Akt1 activity per unit of phosphorylated Akt1 was significantly greater than in PM, indicating that in addition to phosphorylation, another factor(s) modulates Akt1 activation by insulin in rat liver. Our results demonstrate that activation of the insulin receptor kinase and modulation of key components of the insulin signaling cascade occur at the cell surface and within the endosomal system. These data provide further support for the role of the endocytic process in cell signaling. (Endocrinology 141: [4041][4042][4043][4044][4045][4046][4047][4048][4049] 2000)
Endocrinology, 2006
lin-mimicking effects in the rat, selectively activates the endosomal (EN) insulin receptor kinas... more lin-mimicking effects in the rat, selectively activates the endosomal (EN) insulin receptor kinase (IRK) in liver, and markedly abolishes endosomal IRK-associated phosphotyrosine phosphatase (PTP) activity while reducing that of total ENs by approximately 30%. In this study we examined the relatively selective effect of bpv(phen) on endosomal PTP activities for the purpose of defining IRK-associated PTP(s). Using an in-gel PTP assay, we detected multiple (ϳ20) species of endosomal PTP (30 to >220 kDa), with five that were markedly inhibited after in vivo bpV(phen) administration. Using a combination of Mono Q anionic exchange chromatography and immunoblotting, we demonstrated that LAR (leukocyte common antigen-related), PTP-␣, and PTP-1B were present in endosomal subfractions not significantly inhibited by bpv-(phen). PTP-1B activity was assayed in immunoprecipitates from hepatic ENs of control and bpV(phen)-treated rats and was found to be inhibited by approximately 30% after bpv-(phen) treatment. To clarify the role of PTP-1B in dephosphorylating IRK, we prepared hepatic ENs from wild-type and PTP-1B-null mice. We found that the phosphotyrosine content of IRK was similar in these two types of ENs, and that IRK dephosphorylation was not affected in ENs from PTP-1B-null mice compared with that in ENs from wild-type mice. These data suggest that LAR , PTP-␣, and PTP-1B are not candidates for the IRK-associated PTP in hepatic ENs, and that IRK dephosphorylation in ENs may result from the concerted actions of several PTPs.
Quantitative in Vivo Autoradiographic Localization of [ 125 I_Tyr 11 ] Somatostatin-14-and [Leu 8 , d -Trp 22 - 125 ITyr- 125 ] Somatostatin-28-Binding Sites in Rat Brain*
Endocrinology, 1986
Quantitative in vivo autoradiography was used to identify and compare the regional distribution o... more Quantitative in vivo autoradiography was used to identify and compare the regional distribution of specific binding sites for blood-borne [125I-Tyr11]somatostatin-14 (S-14 section) and [Leu8,D-Trp22-125I-Tyr25]somatostatin-28 (S-28 section) in the rat brain. Rats were given intracardiac injections of 17 pmol S-14 section (with or without unlabeled S-14) or 17 pmol S-28 section (with or without unlabeled S-28). After whole body perfusion and fixation, brains were processed for light microscopic autoradiography of S-14 section- and S-28 section-binding sites. Of the peripheral tissues, the adrenal glands showed the highest uptake of S-14 section and S-28 section (determined by counting) and were subsequently processed for autoradiography for comparison with brain. Specific autoradiographic grains (ARG) associated with both radioligands were identified only in the circumventricular organs (CVOs). The highest ARG density associated with S-14 section was found in the area postrema, followed in decreasing order by the subfornical organ and the organum vasculosum lamina terminalis region. Median eminence (ME) contained virtually no specific S-14 section ARG. As with S-14 section, the highest ARG density of S-28 section-binding sites was also found in the area postrema, which labeled approximately equally with the two radioligands. This was followed by the ME, subfornical organ, and organum vasculosum lamina terminalis. The overall patterns of labeling of the CVOs with S-14 section and S-28 section showed significant differences, especially in the ME. Within the ME, labeled S-28 section was concentrated in a broad band throughout the external zone in a location identical to that of immunoreactive S-14. Analysis of dose-response curves obtained with 0.3-30 nmol unlabeled S-14 or S-28 revealed IC50 values for S-14 3- to 6-fold lower than those for S-28 for all labeled CVOs. With both S-14 section and S-28 section, the labelling density of the adrenal glands was double that of the area postrema. Adrenal binding of the radioligands was confined to the cells of the zona glomerulosa. We conclude: specific high affinity binding sites for S-14 section and S-28 section exist in the CVOs and the adrenal glomerulosa; and the 3- to 6-fold higher affinity of binding of S-14 to CVOs compared to S-28 together with the dissimilar patterns of labelling of the different CVOs by the two radioligands suggest the existence of separate populations of S-14 and S-28 receptors.