The CB1 Cannabinoid Receptor: Receptor States, Activity and G Protein Sequestration

Hdl Handle:
http://hdl.handle.net/10675.2/552712
Title:
The CB1 Cannabinoid Receptor: Receptor States, Activity and G Protein Sequestration
Authors:
Nie, Jingjiang
Abstract:
The human CB1 cannabinoid receptor is a member o f the G protein coupled receptor family. The CB1 cannabinoid receptor couples to pertussis toxin sensitive Gi/o proteins and inhibits neuronal voltage-gated Ca2+ channels. The hCBl receptor has two unusual properties: 1) it is constitutively active in the absence o f agonist and 2) it can prevent other G protein coupled receptors from signaling by sequestering a common pool of Gi/o proteins. The mechanism o f constitutive activity and G protein sequestration by the hCB 1 receptor is unknown. In this study, two carboxyl terminal truncation mutants (hCB 1-417 and hCBl-400) were used to test the hypothesis that the proximal carboxyl terminal couples to G proteins while the distal carboxyl terminal modulates G protein sequestration and constitutive activity. Additionally, mutation o f a single amino acid in the second transmembrane domain (rCBl-D164N) was used to test the hypothesis that this amino acid plays a critical role in the structural basis o f G protein coupling and constitutive activity. Receptor cDNA constructs were injected into the nucleus of superior cervical ganglion neurons. After an overnight incubation to allow for receptor expression, neurons were voltage clamped and Ca2+ current were recorded. Inhibition of the Ca2r current by the cannabinoid agonist WIN 55,212-2 was used as an index o f CB1 cannabinoid receptor G protein coupling and activation. Ca2' channels are inhibited by G{3y subunits released from activated Gi/o proteins. In contrast to the wild type CB1 cannabinoid receptor, the mutant receptor in which the entire carboxyl terminal (amino acids 401-472) was deleted (hCB 1-400) failed to inhibit the Ca2+ current. Deletion o f only the distal carboxyl terminal (amino acids 418-472; hCB 1-417) restored Ca2+ current inhibition. These results demonstrate the critical role o f the proximal domain (amino acids 401-417) o f the carboxyl terminal of the hCB 1 receptor in coupling to G proteins. Truncation o f the distal carboxyl terminal domain, however, changed the magnitude o f Ca2+ current inhibition. The hCBl-417 receptor produced significantly less inhibition of the Ca2+ current in the presence o f WIN 55,212-2 compared to the wild type receptor (22.6±3.0% vs 43.7±6.5%, respectively). Thus, the distal carboxyl terminal domain is important in modulating the magnitude of Ca2* current inhibition. In addition to the change in the magnitude o f Ca2+ current inhibition, deletion o f the distal carboxyl terminal significantly slowed the kinetics o f Ca2l~ current inhibition by WIN 55,212-2 (time to peak o f effect: 146.0+8.7 second). The distal carboxyl terminal tail of the CB1 cannabinoid receptor also played a role in constitutive activity and G protein sequestration. The hCB 1-417 receptor displayed enhanced constitutive activity. In neurons injected with 50 ng/pl hCBl-417 cDNA the inverse agonist SR141716A increased the Ca2+ current 101.1±21.3%. The inverse agonist acts to reverse the constitutive activity o f the receptor. The effect o f SRI 41716 A on the hCBl-417 receptor was significantly greater than the 42.9±7.6% Ca2+ current increase in neurons expressing the wild type hCB 1 receptor. G protein sequestration was also enhanced in neurons expressing the truncated hCBl-417 receptor. Wild type hCBl cannabinoid receptors when expressed by injecting 100 ng/p.1 cDNA completely abolish signaling by other G protein coupled receptors including a 2 -adrenergic receptors. Normally activation o f a 2 -adrenergic receptors inhibits the Ca2+ current 44.5±5.7%. In the presence o f hCBl receptors activation of the a 2- adrenergic receptors by UK14304 inhibited the Ca2+ current only 1.5±4.2%. Signaling by a 2-adrenergic receptors can be partially restored by injecting a lower concentration o f hCBl cDNA. In neurons injected with 50 ng/pl hCBl cD N A the ct2 -adrenergic agonist UK14304 inhibited the Ca2+ current 20.0±3.7%. In neurons injected with 50 ng/pl hCBl- 417 cDNA UK14304 inhibited the Ca2+ current 7.0±1.2%. Thus, signaling by the a 2- adrenergic receptor was abolished by the carboxyl terminal truncated hCBl-417 receptor. These results indicate that deletion of the distal carboxyl terminal enhances the ability o f the receptor to sequester G proteins. The aspartic acid residue in the second transmembrane domain of G protein coupled receptors is highly conserved. Mutation o f this aspartic acid (rCBl-D164N) had profound effects on the constitutive activity o f the C B 1 receptor as well as on the ability of the receptor to sequester G proteins. Both the constitutive activity and the ability to sequester G proteins were abolished by the rCBl-D164N receptor. The inverse agonist increased the Ca2+ current only 11.6+6.9% in neurons expressing the mutant rC Bl- D164N receptors. The mutant rCBl-D164N receptors failed to block signaling by the a 2- adrengic receptor. UK14304 inhibited the Ca2+ current 35.8+6.8% in neurons expressing the rCBl-D164N receptors and was not different from control neurons. Additionally, the D164N mutation in the second transmembrane domain decreased the time to peak o f the WIN 55,212-2 inhibition o f the Ca2+ current to 24+4 seconds. These results demonstrate that 1) the proximal carboxyl terminal domain o f the hCB 1 receptor is critical for G protein coupling, 2) the distal carboxyl terminal domain regulates constitutive activity, G protein coupling kinetics and G protein sequestration and 3) the aspartic acid in the second transmembrane domain plays a critical role in G protein sequestration, G protein coupling kinetics and constitutive activity o f the hCBl receptor. Taken together, the mutant receptors shift the CB1 receptor into different receptor states. The D164N-CB1 receptor exists primarily in an inactive state uncoupled from G proteins, the wild type CB1 receptor exists in both active and inactive G protein coupled states and the carboxyl terminal truncated CB 1-417 receptor exists primarily in an active G protein coupled state.
Affiliation:
Not Listed
Issue Date:
2001
URI:
http://hdl.handle.net/10675.2/552712
Additional Links:
http://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/230809129?accountid=12365
Type:
Dissertation
Appears in Collections:
Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorNie, Jingjiangen
dc.date.accessioned2015-05-13T20:34:16Zen
dc.date.available2015-05-13T20:34:16Zen
dc.date.issued2001en
dc.identifier.urihttp://hdl.handle.net/10675.2/552712en
dc.description.abstractThe human CB1 cannabinoid receptor is a member o f the G protein coupled receptor family. The CB1 cannabinoid receptor couples to pertussis toxin sensitive Gi/o proteins and inhibits neuronal voltage-gated Ca2+ channels. The hCBl receptor has two unusual properties: 1) it is constitutively active in the absence o f agonist and 2) it can prevent other G protein coupled receptors from signaling by sequestering a common pool of Gi/o proteins. The mechanism o f constitutive activity and G protein sequestration by the hCB 1 receptor is unknown. In this study, two carboxyl terminal truncation mutants (hCB 1-417 and hCBl-400) were used to test the hypothesis that the proximal carboxyl terminal couples to G proteins while the distal carboxyl terminal modulates G protein sequestration and constitutive activity. Additionally, mutation o f a single amino acid in the second transmembrane domain (rCBl-D164N) was used to test the hypothesis that this amino acid plays a critical role in the structural basis o f G protein coupling and constitutive activity. Receptor cDNA constructs were injected into the nucleus of superior cervical ganglion neurons. After an overnight incubation to allow for receptor expression, neurons were voltage clamped and Ca2+ current were recorded. Inhibition of the Ca2r current by the cannabinoid agonist WIN 55,212-2 was used as an index o f CB1 cannabinoid receptor G protein coupling and activation. Ca2' channels are inhibited by G{3y subunits released from activated Gi/o proteins. In contrast to the wild type CB1 cannabinoid receptor, the mutant receptor in which the entire carboxyl terminal (amino acids 401-472) was deleted (hCB 1-400) failed to inhibit the Ca2+ current. Deletion o f only the distal carboxyl terminal (amino acids 418-472; hCB 1-417) restored Ca2+ current inhibition. These results demonstrate the critical role o f the proximal domain (amino acids 401-417) o f the carboxyl terminal of the hCB 1 receptor in coupling to G proteins. Truncation o f the distal carboxyl terminal domain, however, changed the magnitude o f Ca2+ current inhibition. The hCBl-417 receptor produced significantly less inhibition of the Ca2+ current in the presence o f WIN 55,212-2 compared to the wild type receptor (22.6±3.0% vs 43.7±6.5%, respectively). Thus, the distal carboxyl terminal domain is important in modulating the magnitude of Ca2* current inhibition. In addition to the change in the magnitude o f Ca2+ current inhibition, deletion o f the distal carboxyl terminal significantly slowed the kinetics o f Ca2l~ current inhibition by WIN 55,212-2 (time to peak o f effect: 146.0+8.7 second). The distal carboxyl terminal tail of the CB1 cannabinoid receptor also played a role in constitutive activity and G protein sequestration. The hCB 1-417 receptor displayed enhanced constitutive activity. In neurons injected with 50 ng/pl hCBl-417 cDNA the inverse agonist SR141716A increased the Ca2+ current 101.1±21.3%. The inverse agonist acts to reverse the constitutive activity o f the receptor. The effect o f SRI 41716 A on the hCBl-417 receptor was significantly greater than the 42.9±7.6% Ca2+ current increase in neurons expressing the wild type hCB 1 receptor. G protein sequestration was also enhanced in neurons expressing the truncated hCBl-417 receptor. Wild type hCBl cannabinoid receptors when expressed by injecting 100 ng/p.1 cDNA completely abolish signaling by other G protein coupled receptors including a 2 -adrenergic receptors. Normally activation o f a 2 -adrenergic receptors inhibits the Ca2+ current 44.5±5.7%. In the presence o f hCBl receptors activation of the a 2- adrenergic receptors by UK14304 inhibited the Ca2+ current only 1.5±4.2%. Signaling by a 2-adrenergic receptors can be partially restored by injecting a lower concentration o f hCBl cDNA. In neurons injected with 50 ng/pl hCBl cD N A the ct2 -adrenergic agonist UK14304 inhibited the Ca2+ current 20.0±3.7%. In neurons injected with 50 ng/pl hCBl- 417 cDNA UK14304 inhibited the Ca2+ current 7.0±1.2%. Thus, signaling by the a 2- adrenergic receptor was abolished by the carboxyl terminal truncated hCBl-417 receptor. These results indicate that deletion of the distal carboxyl terminal enhances the ability o f the receptor to sequester G proteins. The aspartic acid residue in the second transmembrane domain of G protein coupled receptors is highly conserved. Mutation o f this aspartic acid (rCBl-D164N) had profound effects on the constitutive activity o f the C B 1 receptor as well as on the ability of the receptor to sequester G proteins. Both the constitutive activity and the ability to sequester G proteins were abolished by the rCBl-D164N receptor. The inverse agonist increased the Ca2+ current only 11.6+6.9% in neurons expressing the mutant rC Bl- D164N receptors. The mutant rCBl-D164N receptors failed to block signaling by the a 2- adrengic receptor. UK14304 inhibited the Ca2+ current 35.8+6.8% in neurons expressing the rCBl-D164N receptors and was not different from control neurons. Additionally, the D164N mutation in the second transmembrane domain decreased the time to peak o f the WIN 55,212-2 inhibition o f the Ca2+ current to 24+4 seconds. These results demonstrate that 1) the proximal carboxyl terminal domain o f the hCB 1 receptor is critical for G protein coupling, 2) the distal carboxyl terminal domain regulates constitutive activity, G protein coupling kinetics and G protein sequestration and 3) the aspartic acid in the second transmembrane domain plays a critical role in G protein sequestration, G protein coupling kinetics and constitutive activity o f the hCBl receptor. Taken together, the mutant receptors shift the CB1 receptor into different receptor states. The D164N-CB1 receptor exists primarily in an inactive state uncoupled from G proteins, the wild type CB1 receptor exists in both active and inactive G protein coupled states and the carboxyl terminal truncated CB 1-417 receptor exists primarily in an active G protein coupled state.en
dc.relation.urlhttp://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/230809129?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.subjectCannabinoid Receptoren
dc.subjectCa2+ Channelsen
dc.subjectG protein coupled receptorsen
dc.subjectSympathetic Neuronen
dc.subjectWIN 55,23 12-2en
dc.subjectSR 141716Aen
dc.titleThe CB1 Cannabinoid Receptor: Receptor States, Activity and G Protein Sequestrationen
dc.typeDissertationen
dc.contributor.departmentNot Listeden
dc.description.advisorLewis, Deborah L.en
dc.description.committeeGanapathy, Vadivel; Creazzo, Tony; Bergson, Clare; Lambert, Nevinen
dc.description.degreeDoctor of Philosophy (Ph.D.)en
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