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dc.contributor.authorLiu, Yunguang
dc.date.accessioned2015-04-06T18:58:40Zen
dc.date.available2015-04-06T18:58:40Zen
dc.date.issued2006-10en
dc.identifier.urihttp://hdl.handle.net/10675.2/348546
dc.description.abstractCBi receptors modulate synaptic transmission and play important roles in analgesia, appetite and neuroprotection. However, little is known about how CBi activity is regulated. The possibility that a novel protein CRIPla interacts with CBj was studied to determine whether CBt functions are modulated by such an interaction. CRIPla specifically interacted with the C-terminus of CBi in a GST pull-down assay and co-precipitated with CBi oligomer in HEK293 cells, demonstrating that CRIPla interacts with CB^ Moreover, CRIPla and CBj co-localized when heterologously expressed in HEK293 cells and in rat superior cervical ganglion (SCG) neurons. The functional CBr CRIPla interaction was investigated using whole-cell voltage-clamp recordings of N-type Ca2+ channels in SCG neurons heterologously expressing CBi with or without CRIPla. The electrophysiological data demonstrated that CRIPla significantly reduced the ability of the CB! inverse agonist SR141716 to enhance the Ca2+ current but did not affect the ability of the CBi agonist WIN 55,212- 2 to inhibit the Ca2+ current. In addition, CRIPla significantly decreased Ca2+ current basal facilitation ratio. Since CRIPla did not alter CB! expression pattern or the EC50 response to WIN 55,212-2 in neurons co-expressing CRIPla and CBi; it is unlikely that the attenuated SR141716 response or the reduced Ca2+ current basal facilitation ratio resulted from decreased surface expression of CB,. Our data indicate that CRIPla inhibits the constitutive activity of CB! receptors. Deletion of the last nine amino acids of the CBi receptor abolished the interaction with CRIPla in a GST pull-down assay, indicating that these residues of the CBi receptor constitute the CRIPla interaction domain. In SCG neurons, the CBiA465-473 receptor missing the CRIPla interaction domain was constitutively active but the constitutive activity was not affected by CRIPla, demonstrating that deletion of the CRIPla interaction domain from CB! receptors reversed the ability of CRIPla to inhibit CBi constitutive activity. Taken together, our data suggest that CRIPla controls CBi constitutive activity by interacting with the last nine amino acids of the CBi receptor.
dc.relation.urlhttp://search.proquest.com/docview/304954147?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectCB1 cannabinoid receptoren
dc.subjectInteracting proteinen
dc.subjectCR1P1aen
dc.subjectConstitutive activityen
dc.subjectCa2+ Channelsen
dc.subjectInteraction domainen
dc.titleCharacterization of the Physical and Functional Interaction between a Novel Protein CRIPla and the CBj Cannabinoid Receptoren
dc.typeDissertationen
dc.contributor.departmentDepartment of Pharmacology and Toxicologyen
dc.description.advisorLewis, Deborah L.en
dc.description.degreeDoctor of Philosophy (Ph.D.)en
dc.description.committeeBergson, Clare; Lambert, Nevin; Redmond, Lori; Terry, Alvin; Lious, Gregory; Barman, Scotten
html.description.abstractCBi receptors modulate synaptic transmission and play important roles in analgesia, appetite and neuroprotection. However, little is known about how CBi activity is regulated. The possibility that a novel protein CRIPla interacts with CBj was studied to determine whether CBt functions are modulated by such an interaction. CRIPla specifically interacted with the C-terminus of CBi in a GST pull-down assay and co-precipitated with CBi oligomer in HEK293 cells, demonstrating that CRIPla interacts with CB^ Moreover, CRIPla and CBj co-localized when heterologously expressed in HEK293 cells and in rat superior cervical ganglion (SCG) neurons. The functional CBr CRIPla interaction was investigated using whole-cell voltage-clamp recordings of N-type Ca2+ channels in SCG neurons heterologously expressing CBi with or without CRIPla. The electrophysiological data demonstrated that CRIPla significantly reduced the ability of the CB! inverse agonist SR141716 to enhance the Ca2+ current but did not affect the ability of the CBi agonist WIN 55,212- 2 to inhibit the Ca2+ current. In addition, CRIPla significantly decreased Ca2+ current basal facilitation ratio. Since CRIPla did not alter CB! expression pattern or the EC50 response to WIN 55,212-2 in neurons co-expressing CRIPla and CBi; it is unlikely that the attenuated SR141716 response or the reduced Ca2+ current basal facilitation ratio resulted from decreased surface expression of CB,. Our data indicate that CRIPla inhibits the constitutive activity of CB! receptors. Deletion of the last nine amino acids of the CBi receptor abolished the interaction with CRIPla in a GST pull-down assay, indicating that these residues of the CBi receptor constitute the CRIPla interaction domain. In SCG neurons, the CBiA465-473 receptor missing the CRIPla interaction domain was constitutively active but the constitutive activity was not affected by CRIPla, demonstrating that deletion of the CRIPla interaction domain from CB! receptors reversed the ability of CRIPla to inhibit CBi constitutive activity. Taken together, our data suggest that CRIPla controls CBi constitutive activity by interacting with the last nine amino acids of the CBi receptor.


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