title: Cannabinoid Receptor Interacting Protein 1a Competition with β-Arrestin for CB1 Receptor Binding Sites.
abstract: Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1receptor (CB1R) distal C-terminal-associated protein that alters CB1R interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CB1R interactions with β-arrestin proteins that interact with the CB1R at the C-terminus. Coimmunoprecipitation studies indicated that CB1R associates in complexes with either CRIP1a or β-arrestin, but CRIP1a and β-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and β-arrestin binding to the CB1R, which we hypothesized could attenuate the action of β-arrestin to mediate CB1R internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CB1Rs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CB1R. CRIP1a overexpression attenuated CP55940-mediated GFP-CB1R as well as endogenous β-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented β-arrestin redistribution to punctae. Peptides mimicking the CB1R C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CB1R distal C-terminus 14-mer peptide reduced CB1R-CRIP1a association. Coimmunoprecipitation of CB1R protein complexes demonstrated that central or distal C-terminal peptides competed for the CB1R association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with β-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with β-arrestin 2. Thus, CRIP1a can compete with β-arrestins for interaction with C-terminal CB1R domains that could affect agonist-driven, β-arrestin-mediated internalization of the CB1R.
authors.affiliation: Department of Physiology and Pharmacology (L.C.B., T.P, K.E., S.L.-K., A.A.I., B.M.K., J.E.O., C.E.B., A.C.H.) and Department of Biochemistry and Center for Structural Biology (R.R.H., W.T.L.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; Department of Chemistry (T.P.) and Center for Molecular Signaling (W.T.L., A.C.H.), Wake Forest University, Winston-Salem, North Carolina; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (D.E.S.); and AL Azhar Faculty of Medicine, New Damietta, Egypt (K.E.).
authors.forename: Lawrence C
authors.initials: LC
authors.lastname: Blume
authors.affiliation: Department of Physiology and Pharmacology (L.C.B., T.P, K.E., S.L.-K., A.A.I., B.M.K., J.E.O., C.E.B., A.C.H.) and Department of Biochemistry and Center for Structural Biology (R.R.H., W.T.L.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; Department of Chemistry (T.P.) and Center for Molecular Signaling (W.T.L., A.C.H.), Wake Forest University, Winston-Salem, North Carolina; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (D.E.S.); and AL Azhar Faculty of Medicine, New Damietta, Egypt (K.E.).
authors.forename: Theresa
authors.initials: T
authors.lastname: Patten
authors.affiliation: Department of Physiology and Pharmacology (L.C.B., T.P, K.E., S.L.-K., A.A.I., B.M.K., J.E.O., C.E.B., A.C.H.) and Department of Biochemistry and Center for Structural Biology (R.R.H., W.T.L.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; Department of Chemistry (T.P.) and Center for Molecular Signaling (W.T.L., A.C.H.), Wake Forest University, Winston-Salem, North Carolina; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (D.E.S.); and AL Azhar Faculty of Medicine, New Damietta, Egypt (K.E.).
authors.forename: Khalil
authors.initials: K
authors.lastname: Eldeeb
authors.affiliation: Department of Physiology and Pharmacology (L.C.B., T.P, K.E., S.L.-K., A.A.I., B.M.K., J.E.O., C.E.B., A.C.H.) and Department of Biochemistry and Center for Structural Biology (R.R.H., W.T.L.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; Department of Chemistry (T.P.) and Center for Molecular Signaling (W.T.L., A.C.H.), Wake Forest University, Winston-Salem, North Carolina; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (D.E.S.); and AL Azhar Faculty of Medicine, New Damietta, Egypt (K.E.).
authors.forename: Sandra
authors.initials: S
authors.lastname: Leone-Kabler
authors.affiliation: Department of Physiology and Pharmacology (L.C.B., T.P, K.E., S.L.-K., A.A.I., B.M.K., J.E.O., C.E.B., A.C.H.) and Department of Biochemistry and Center for Structural Biology (R.R.H., W.T.L.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; Department of Chemistry (T.P.) and Center for Molecular Signaling (W.T.L., A.C.H.), Wake Forest University, Winston-Salem, North Carolina; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (D.E.S.); and AL Azhar Faculty of Medicine, New Damietta, Egypt (K.E.).
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Ilyasov
authors.affiliation: Department of Physiology and Pharmacology (L.C.B., T.P, K.E., S.L.-K., A.A.I., B.M.K., J.E.O., C.E.B., A.C.H.) and Department of Biochemistry and Center for Structural Biology (R.R.H., W.T.L.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; Department of Chemistry (T.P.) and Center for Molecular Signaling (W.T.L., A.C.H.), Wake Forest University, Winston-Salem, North Carolina; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (D.E.S.); and AL Azhar Faculty of Medicine, New Damietta, Egypt (K.E.).
authors.forename: Bradley M
authors.initials: BM
authors.lastname: Keegan
authors.affiliation: Department of Physiology and Pharmacology (L.C.B., T.P, K.E., S.L.-K., A.A.I., B.M.K., J.E.O., C.E.B., A.C.H.) and Department of Biochemistry and Center for Structural Biology (R.R.H., W.T.L.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; Department of Chemistry (T.P.) and Center for Molecular Signaling (W.T.L., A.C.H.), Wake Forest University, Winston-Salem, North Carolina; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (D.E.S.); and AL Azhar Faculty of Medicine, New Damietta, Egypt (K.E.).
authors.forename: Jeremy E
authors.initials: JE
authors.lastname: O'Neal
authors.affiliation: Department of Physiology and Pharmacology (L.C.B., T.P, K.E., S.L.-K., A.A.I., B.M.K., J.E.O., C.E.B., A.C.H.) and Department of Biochemistry and Center for Structural Biology (R.R.H., W.T.L.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; Department of Chemistry (T.P.) and Center for Molecular Signaling (W.T.L., A.C.H.), Wake Forest University, Winston-Salem, North Carolina; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (D.E.S.); and AL Azhar Faculty of Medicine, New Damietta, Egypt (K.E.).
authors.forename: Caroline E
authors.initials: CE
authors.lastname: Bass
authors.affiliation: Department of Physiology and Pharmacology (L.C.B., T.P, K.E., S.L.-K., A.A.I., B.M.K., J.E.O., C.E.B., A.C.H.) and Department of Biochemistry and Center for Structural Biology (R.R.H., W.T.L.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; Department of Chemistry (T.P.) and Center for Molecular Signaling (W.T.L., A.C.H.), Wake Forest University, Winston-Salem, North Carolina; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (D.E.S.); and AL Azhar Faculty of Medicine, New Damietta, Egypt (K.E.).
authors.forename: Roy R
authors.initials: RR
authors.lastname: Hantgan
authors.affiliation: Department of Physiology and Pharmacology (L.C.B., T.P, K.E., S.L.-K., A.A.I., B.M.K., J.E.O., C.E.B., A.C.H.) and Department of Biochemistry and Center for Structural Biology (R.R.H., W.T.L.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; Department of Chemistry (T.P.) and Center for Molecular Signaling (W.T.L., A.C.H.), Wake Forest University, Winston-Salem, North Carolina; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (D.E.S.); and AL Azhar Faculty of Medicine, New Damietta, Egypt (K.E.).
authors.forename: W Todd
authors.initials: WT
authors.lastname: Lowther
authors.affiliation: Department of Physiology and Pharmacology (L.C.B., T.P, K.E., S.L.-K., A.A.I., B.M.K., J.E.O., C.E.B., A.C.H.) and Department of Biochemistry and Center for Structural Biology (R.R.H., W.T.L.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; Department of Chemistry (T.P.) and Center for Molecular Signaling (W.T.L., A.C.H.), Wake Forest University, Winston-Salem, North Carolina; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (D.E.S.); and AL Azhar Faculty of Medicine, New Damietta, Egypt (K.E.).
authors.forename: Dana E
authors.initials: DE
authors.lastname: Selley
authors.affiliation: Department of Physiology and Pharmacology (L.C.B., T.P, K.E., S.L.-K., A.A.I., B.M.K., J.E.O., C.E.B., A.C.H.) and Department of Biochemistry and Center for Structural Biology (R.R.H., W.T.L.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; Department of Chemistry (T.P.) and Center for Molecular Signaling (W.T.L., A.C.H.), Wake Forest University, Winston-Salem, North Carolina; Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (D.E.S.); and AL Azhar Faculty of Medicine, New Damietta, Egypt (K.E.) ahowlett@wakehealth.edu.
authors.forename: A Llyn C
authors.initials: AL
authors.lastname: Howlett
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000378:metabolism*; D000071557:beta-Arrestins / Q000378:metabolism*
chemical_list: C000618264:CNRIP1 protein, human; D002352:Carrier Proteins; C540799:Cnrip1 protein, rat; D008565:Membrane Proteins; D010455:Peptides; D043884:Receptor, Cannabinoid, CB1; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins