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Functional and molecular evidence of B3-adrenoceptors in smooth muscle cells isolated from the longitudinal and circular muscle layers of human colon
April 2000
system. There are only few studies that describe the expression of CCK receptors on human leukemia-derived cell lines but the receptor structure and function in normal PBMC have not been clearly established. AIM: To determine CCK receptor expression, structure, and function in human peripheral blood mononuclear cells. METHODS: PBMC were isolated from peripheral blood of healthy donors bi Ficoll density gradient centrifugation. RNA was extracted from 2 x 10 cells using standard protocols. First strand cDNA synthesis was accomplished with 4-5 JLg of total RNA. Six hundred to 800 ng cDNA were utilized for amplification by PCR using CCK-A and CCK-B receptor specific oligonucleotide primers. PCR products were subcloned into the expression vector pCR 2.1 and subjected to dideoxy sequencing in full length. RESULTS: Full-length cDNA clones encoding the human CCK-A and CCK-B/gastrin receptor are expressed in PBMC from healthy volunteers without hematopoietic malignancy. Sequence comparisons with deposited data bank entries revealed that the cloned CCK receptors subtypes are wild type sequence and identical to the cDNAs isolated from gallbladder and stomach. Besides wild type fulllength cDNA clones encoding the CCK-B/gastrin receptor, two CCK-B/ gastrin receptor splice variants were isolated. CCK-BR cDNA splice variant A spans exons I-V but retains intron IV. Variant Blacks exon IV completely. In vitro, gastrin decreased 3H-thymidine labeling in PHApretreated [2 JLg/ml] PBMC at a half-maximally effective concentration of 1,5 nM. CONCLUSION: CCK-A and CCK-B-receptor mRNA are expressed in human PBMC. Functional characterization of CCK receptors in PBMC reveals that gastrin exhibits antiproliferative effects in PHA-stimulated PBMC. The physiological significance of splice variants of the CCK-B/gastrin receptor in PBMC remains to be elucidated.
AGAA309
membrane. HEK 293 cells express a previously uncharacterized endogenous VIP responsive receptor which could be useful in the study of receptor regulation. METHODS: We used primers specifically designed to discriminate between the human VIP type I and VIP type 2 receptors and RT-PCR to determine the VIP receptor expressed endogenously by HEK 293 cells. To study endogenous receptor signaling and desensitization, HEK 293 cells expressing the endogenous VIP receptor were transfected with various amounts of cDNA for GRKs 4, 5, and 6 and receptor signaling and desensitization was quantitiated. Agonist stimulated receptor signaling was assessed by cAMP accumulation. Increasing amounts of GRK expression were determined by western blotting using GRK specific antisera. RESULTS: HEK 293 cells endogenously express the VIP type I receptor. Transient transfection with increasing amounts of GRK 5 and 6 led to GRK-dependent desensitization. However, transfection with various amounts of GRK 4 eDNA did not alter VIP receptor signaling as determined by cAMP accumulation. Western blotting using GRK specific antisera showed that with increasing expression of GRKs 5 and 6, cAMP accumulation decreased accordingly, demonstrating a dose dependent effect. CONCLUSION: HEK 293 cells endogenously express the type I VIP receptor. This receptor can be used to study receptor regulation by GRKs and potentially other signal transduction molecules. The endogenous VIP type I receptor can be regulated by GRK 5 and 6. Transient transfection of increasing amounts of cDNA for GRKs 5 and 6 produces increasing amounts of protein expression assessed by western blotting. Studies of endogenously expressed receptors should improve and expand our understanding of receptor regulation. 1705
1703 FUNCTIONAL AND MOLECULAR EVIDENCE OF B3-ADRENOCEPTORS IN SMOOTH MUSCLE CELLS ISOLATED FROM THE LONGITUDINAL AND CIRCULAR MUSCLE LAYERS OF HUMAN COLON. Carola Severi, Giovanna Romano, Vito D. Corleto, Tiziano Croci, Gianfranco Delle Fave, Univ La Sapienza, Roma, Italy; Research Ctr SANOFI Midy, Milano, Italy. A population of atypical f3-adrenoceptors, similar to the f3radrenoceptors of adypocites, has been reported in various tissues including human colon where they appear to mediate relaxation of circular and longitudinal smooth muscle. The relative myogenic and neural contributions to this effect need however to be clearly investigated. Aim of the study was to evaluate the direct relaxant effect of two f33-agonists, CGPI2177A and SR58611A, on smooth muscle cells (SMC) isolated from longitudinal and circular smooth muscle layers of human colon and to compare their effects with the relaxation induced by (-)isoproterenol. f33-adrenoceptors expression was also determined on both layers by RT-PCR. Relaxation is expressed as percentage of inhibition of maximal contraction induced by carbachol (30nM). Longitudinal and circular SMC presented similar resting cell length (86.7::'::0.9 and 86.0::'::0.2JLm respectively) which decreased in both cells by 20% in response to carbachol (maximal contraction). (-)Isoproterenol induced a 63.4::'::3.0% and a 55.2::':: 1.7% maximal relaxation of longitudinal and circular SMC respectively, with equal Crn ax of 0.1 mM and similar ICso (2 and I JLM). The f31-antagonist CGP20712A and the f32-antagonist ICI118551, both at I JLM, had no effect on isoproterenol ICso but reduced of 30% the agonist maximal effect in both layers. The f33-adrenoceptor agonists CGPI2177A and SR5861lA induced a similar degree of relaxation as (-)isoproterenol in the presence of the {3,- and the f3rantagonists. On longitudinal SMC, CGPI2177A induced a maximal relaxation (Cma x O.lmM) amounting to 75::'::3% of the response with (-)isoproterenol alone with an ICso of 0.4 JLM and on circular SMC (C rn a , O.lmM)a maximal relaxation of 83.2::'::2.5% of the response to isoproterenol with an ICso of 1.2 JLM. SR58611A (Cmax 0.1mM)induced a 82.0::'::5.0% and a 85.5::'::5.1 % maximal relaxation of the maximal response to (-)isoproterenol alone on longitudinal and circular SMC respectively with similar ICso (Long: 1.6 JLM; Circ: 1.1 JLM). RT-PCR of total human circular or longitudinal colonic SMC RNA yielded detectable expression of f33-adrenoceptors in both layers. In conclusion, the scant activity of the f3,and f32- antagonists against (-)isoproterenol response, the relaxation induced by the f33-agonists CGPI2177A and SR58611A in longitudinal and circular SMC and the expression of the f33-adrenoceptors in both layers strongly support a functional role of the f33-adrenoceptor in human colon.
1704 G PROTEIN-COUPLED RECEPTOR KINASE (GRK) SPECIFICITY OF ENDOGENOUS TYPE 1 VASOACTIVE INTESTINAL POLVPEPTIDE (VIP) RECEPTOR EXPRESSED ON THE SURFACE OF HEK 293 CELLS. Michael A. Shetzline, Julia K. Walker, Brian M. Curtin, Richard T. Premont, Marc G. Caron, Duke Univ Med Ctr, Durham, NC. INTRODUCTION: G protein-coupled receptor (GPCR) regulation is routinely studied by overexpression of receptor constructs in transfected cell lines. Using this method the role of GRKs and f3-arrestin in receptor desensitization has been established for many GPCRs. However, endogenous receptor regulation is much less studied. Difficulties arise in defining the receptor type expressed and getting sufficient expression to study regulatory mechanisms. We previously demonstrated GRK specific regulation of the VIP receptor overexpressed in HEK 293 cells, as well as VIP-dependent f3-arrestin translocation from the cytosol to the plasma
THE ROLE OF PROTEIN KINASE A (PKA) DEPENDENT PHOSPHORYLATION OF THE SECRETIN RECEPTOR IN RECEPTOR INTERNALIZATION. Michael A. Shetzline, Julia K. Walker, Brian M. Curtin, Richard T. Premont, Marc G. Caron, Duke Univ Med Ctr, Durham, NC. G protein-coupled receptor (GPCR) phosphorylation is known to be important for receptor internalization. We have demonstrated that the secretin receptor, a member of class Il GPCRs, may use a pathway for receptor internalization which differs from that of other GPCRs (ffiC,274: 31515,1999). Agonist-stimulated secretin receptor internalization is inhibited by incubation with H-89 and staurosporin. These PKA inhibitors may exert their effect on receptor internalization by preventing secretin receptor phosphorylation directly or by inhibiting the phosphorylation of substrates involved in secretin receptor internalization or trafficking. METHODS: To investigate the role of PKA dependent phosphorylation of the secretin receptor, we used site-directed mutagenesis to produce secretin receptor constructs void of potential PKA phosphorylation sites (S31OA, S409A, S31O/409A).Mutagenesis was confirmed by sequencing. These constructs contain an N-terminal FLAG epitope which allows for fluorescent labeling. HEK 293 cells were transiently transfected with wild-type and mutant receptors. Dose-response receptor signaling was quantitated for all constructs by cAMP accumulation. Receptor internalization was assessed after 30 minutes of agonist exposure by fluorescent-activated cell sorting (FACS) and reported as loss of cell surface receptors. RESULTS: Substitution of alanine for serine at potential PKA phosphorylation sites of the secretin receptor did not alter receptor signaling (similar ECso for cAMP accumulation). However, these mutants demonstrated reduced receptor internalization when exposed to agonist for 30 minutes assessed by FACS analysis and loss of cell surface receptors. The S310A mutation reduced secretin receptor internalization 20 +/- 5%; S409A 33 +/- 8%; and S31O/409A 36 +/- 12%. CONCLUSIONS: Here we demonstrate that removal of potential PKA phosphorylation sites in the secretin receptor reduces secretin receptor internalization. This is in contrast to the f32adrenergic receptor, which does not demonstrate reduced receptor internalization upon removal of known PKA phosphorylation sites. However, the PKA phosphorylation deficient mutant is still able to be internalized and this may imply a role for PKA dependent phosphorylation of other substrates necessary for secretin receptor internalization. Also, the secretin receptor may contain sites other than those mutated here which can be phosphorylated by PKA and are important for its internalization.
system. There are only few studies that describe the expression of CCK receptors on human leukemia-derived cell lines but the receptor structure and function in normal PBMC have not been clearly established. AIM: To determine CCK receptor expression, structure, and function in human peripheral blood mononuclear cells. METHODS: PBMC were isolated from peripheral blood of healthy donors bi Ficoll density gradient centrifugation. RNA was extracted from 2 x 10 cells using standard protocols. First strand cDNA synthesis was accomplished with 4-5 JLg of total RNA. Six hundred to 800 ng cDNA were utilized for amplification by PCR using CCK-A and CCK-B receptor specific oligonucleotide primers. PCR products were subcloned into the expression vector pCR 2.1 and subjected to dideoxy sequencing in full length. RESULTS: Full-length cDNA clones encoding the human CCK-A and CCK-B/gastrin receptor are expressed in PBMC from healthy volunteers without hematopoietic malignancy. Sequence comparisons with deposited data bank entries revealed that the cloned CCK receptors subtypes are wild type sequence and identical to the cDNAs isolated from gallbladder and stomach. Besides wild type fulllength cDNA clones encoding the CCK-B/gastrin receptor, two CCK-B/ gastrin receptor splice variants were isolated. CCK-BR cDNA splice variant A spans exons I-V but retains intron IV. Variant Blacks exon IV completely. In vitro, gastrin decreased 3H-thymidine labeling in PHApretreated [2 JLg/ml] PBMC at a half-maximally effective concentration of 1,5 nM. CONCLUSION: CCK-A and CCK-B-receptor mRNA are expressed in human PBMC. Functional characterization of CCK receptors in PBMC reveals that gastrin exhibits antiproliferative effects in PHA-stimulated PBMC. The physiological significance of splice variants of the CCK-B/gastrin receptor in PBMC remains to be elucidated.
AGAA309
membrane. HEK 293 cells express a previously uncharacterized endogenous VIP responsive receptor which could be useful in the study of receptor regulation. METHODS: We used primers specifically designed to discriminate between the human VIP type I and VIP type 2 receptors and RT-PCR to determine the VIP receptor expressed endogenously by HEK 293 cells. To study endogenous receptor signaling and desensitization, HEK 293 cells expressing the endogenous VIP receptor were transfected with various amounts of cDNA for GRKs 4, 5, and 6 and receptor signaling and desensitization was quantitiated. Agonist stimulated receptor signaling was assessed by cAMP accumulation. Increasing amounts of GRK expression were determined by western blotting using GRK specific antisera. RESULTS: HEK 293 cells endogenously express the VIP type I receptor. Transient transfection with increasing amounts of GRK 5 and 6 led to GRK-dependent desensitization. However, transfection with various amounts of GRK 4 eDNA did not alter VIP receptor signaling as determined by cAMP accumulation. Western blotting using GRK specific antisera showed that with increasing expression of GRKs 5 and 6, cAMP accumulation decreased accordingly, demonstrating a dose dependent effect. CONCLUSION: HEK 293 cells endogenously express the type I VIP receptor. This receptor can be used to study receptor regulation by GRKs and potentially other signal transduction molecules. The endogenous VIP type I receptor can be regulated by GRK 5 and 6. Transient transfection of increasing amounts of cDNA for GRKs 5 and 6 produces increasing amounts of protein expression assessed by western blotting. Studies of endogenously expressed receptors should improve and expand our understanding of receptor regulation. 1705
1703 FUNCTIONAL AND MOLECULAR EVIDENCE OF B3-ADRENOCEPTORS IN SMOOTH MUSCLE CELLS ISOLATED FROM THE LONGITUDINAL AND CIRCULAR MUSCLE LAYERS OF HUMAN COLON. Carola Severi, Giovanna Romano, Vito D. Corleto, Tiziano Croci, Gianfranco Delle Fave, Univ La Sapienza, Roma, Italy; Research Ctr SANOFI Midy, Milano, Italy. A population of atypical f3-adrenoceptors, similar to the f3radrenoceptors of adypocites, has been reported in various tissues including human colon where they appear to mediate relaxation of circular and longitudinal smooth muscle. The relative myogenic and neural contributions to this effect need however to be clearly investigated. Aim of the study was to evaluate the direct relaxant effect of two f33-agonists, CGPI2177A and SR58611A, on smooth muscle cells (SMC) isolated from longitudinal and circular smooth muscle layers of human colon and to compare their effects with the relaxation induced by (-)isoproterenol. f33-adrenoceptors expression was also determined on both layers by RT-PCR. Relaxation is expressed as percentage of inhibition of maximal contraction induced by carbachol (30nM). Longitudinal and circular SMC presented similar resting cell length (86.7::'::0.9 and 86.0::'::0.2JLm respectively) which decreased in both cells by 20% in response to carbachol (maximal contraction). (-)Isoproterenol induced a 63.4::'::3.0% and a 55.2::':: 1.7% maximal relaxation of longitudinal and circular SMC respectively, with equal Crn ax of 0.1 mM and similar ICso (2 and I JLM). The f31-antagonist CGP20712A and the f32-antagonist ICI118551, both at I JLM, had no effect on isoproterenol ICso but reduced of 30% the agonist maximal effect in both layers. The f33-adrenoceptor agonists CGPI2177A and SR5861lA induced a similar degree of relaxation as (-)isoproterenol in the presence of the {3,- and the f3rantagonists. On longitudinal SMC, CGPI2177A induced a maximal relaxation (Cma x O.lmM) amounting to 75::'::3% of the response with (-)isoproterenol alone with an ICso of 0.4 JLM and on circular SMC (C rn a , O.lmM)a maximal relaxation of 83.2::'::2.5% of the response to isoproterenol with an ICso of 1.2 JLM. SR58611A (Cmax 0.1mM)induced a 82.0::'::5.0% and a 85.5::'::5.1 % maximal relaxation of the maximal response to (-)isoproterenol alone on longitudinal and circular SMC respectively with similar ICso (Long: 1.6 JLM; Circ: 1.1 JLM). RT-PCR of total human circular or longitudinal colonic SMC RNA yielded detectable expression of f33-adrenoceptors in both layers. In conclusion, the scant activity of the f3,and f32- antagonists against (-)isoproterenol response, the relaxation induced by the f33-agonists CGPI2177A and SR58611A in longitudinal and circular SMC and the expression of the f33-adrenoceptors in both layers strongly support a functional role of the f33-adrenoceptor in human colon.
1704 G PROTEIN-COUPLED RECEPTOR KINASE (GRK) SPECIFICITY OF ENDOGENOUS TYPE 1 VASOACTIVE INTESTINAL POLVPEPTIDE (VIP) RECEPTOR EXPRESSED ON THE SURFACE OF HEK 293 CELLS. Michael A. Shetzline, Julia K. Walker, Brian M. Curtin, Richard T. Premont, Marc G. Caron, Duke Univ Med Ctr, Durham, NC. INTRODUCTION: G protein-coupled receptor (GPCR) regulation is routinely studied by overexpression of receptor constructs in transfected cell lines. Using this method the role of GRKs and f3-arrestin in receptor desensitization has been established for many GPCRs. However, endogenous receptor regulation is much less studied. Difficulties arise in defining the receptor type expressed and getting sufficient expression to study regulatory mechanisms. We previously demonstrated GRK specific regulation of the VIP receptor overexpressed in HEK 293 cells, as well as VIP-dependent f3-arrestin translocation from the cytosol to the plasma
THE ROLE OF PROTEIN KINASE A (PKA) DEPENDENT PHOSPHORYLATION OF THE SECRETIN RECEPTOR IN RECEPTOR INTERNALIZATION. Michael A. Shetzline, Julia K. Walker, Brian M. Curtin, Richard T. Premont, Marc G. Caron, Duke Univ Med Ctr, Durham, NC. G protein-coupled receptor (GPCR) phosphorylation is known to be important for receptor internalization. We have demonstrated that the secretin receptor, a member of class Il GPCRs, may use a pathway for receptor internalization which differs from that of other GPCRs (ffiC,274: 31515,1999). Agonist-stimulated secretin receptor internalization is inhibited by incubation with H-89 and staurosporin. These PKA inhibitors may exert their effect on receptor internalization by preventing secretin receptor phosphorylation directly or by inhibiting the phosphorylation of substrates involved in secretin receptor internalization or trafficking. METHODS: To investigate the role of PKA dependent phosphorylation of the secretin receptor, we used site-directed mutagenesis to produce secretin receptor constructs void of potential PKA phosphorylation sites (S31OA, S409A, S31O/409A).Mutagenesis was confirmed by sequencing. These constructs contain an N-terminal FLAG epitope which allows for fluorescent labeling. HEK 293 cells were transiently transfected with wild-type and mutant receptors. Dose-response receptor signaling was quantitated for all constructs by cAMP accumulation. Receptor internalization was assessed after 30 minutes of agonist exposure by fluorescent-activated cell sorting (FACS) and reported as loss of cell surface receptors. RESULTS: Substitution of alanine for serine at potential PKA phosphorylation sites of the secretin receptor did not alter receptor signaling (similar ECso for cAMP accumulation). However, these mutants demonstrated reduced receptor internalization when exposed to agonist for 30 minutes assessed by FACS analysis and loss of cell surface receptors. The S310A mutation reduced secretin receptor internalization 20 +/- 5%; S409A 33 +/- 8%; and S31O/409A 36 +/- 12%. CONCLUSIONS: Here we demonstrate that removal of potential PKA phosphorylation sites in the secretin receptor reduces secretin receptor internalization. This is in contrast to the f32adrenergic receptor, which does not demonstrate reduced receptor internalization upon removal of known PKA phosphorylation sites. However, the PKA phosphorylation deficient mutant is still able to be internalized and this may imply a role for PKA dependent phosphorylation of other substrates necessary for secretin receptor internalization. Also, the secretin receptor may contain sites other than those mutated here which can be phosphorylated by PKA and are important for its internalization.