The Carboxyl Terminus of the Prolactin-Releasing Peptide Receptor Interacts with PDZ Domain Proteins Involved in α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid Receptor Clustering
- Departments of 1Pharmacology (S.H.S.L., Z.W., H.-P.N., O.C.) and2Developmental and Cellular Biology (O.C.), College of Medicine, University of California, Irvine, California; 3Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois (A.C.A.); and 4NeoGene Technologies, Inc., Irvine, California (Z.W., H.-P.W., O.C.)
- Dr. Oliver Civelli, Department of Pharmacology, 354 Medical Surge II, University of California, Irvine, Irvine, CA 92697. E-mail: ocivelli{at}uci.edu
Abstract
PDZ domain proteins use the PDZ domain binding motif to bind to the C-terminal sequence of membrane proteins to help scaffold them and spatially organize the components of the intracellular signaling machinery. We have identified a sequence at the C terminus of a G protein-coupled receptor, the PrRP receptor, that shares similarities with the C-terminal sequence of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPA-R) subunits that interact with PDZ domain proteins. When coexpressed in human embryonic kidney 293 cells, PrRP receptor was able to coimmunoprecipitate the three PDZ domain proteins known to interact with AMPA receptors: glutamate receptor interacting protein (GRIP), AMPA binding protein (ABP), and protein that interacts with C-kinase (PICK1), but not the PDZ domain protein PSD-95, which does not interact with AMPA receptors. These interactions are sequence-selective as determined by mutagenesis. Furthermore, we show that PrRP receptor forms intracellular clusters when coexpressed with PICK1, and that this clustering effect is dependent on the interaction between the PICK1 PDZ domain and the last four amino acids of PrRP receptor. We found that PrRP receptor interaction with GRIP is not protein kinase C-regulated but may be regulated by other unidentified kinase because okadaic acid dramatically reduced GRIP interaction. By in situ hybridization, we show that the PrRP receptor is expressed in neurons that also express these PDZ domain proteins. We thus demonstrate that PrRP receptor interacts with the same PDZ domain proteins as the AMPA-Rs, raising the possibility that these two proteins could be scaffolded together at the synapse. These results may help to gain important insights into PrRP functions within the central nervous system.
Footnotes
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This work was supported in part by a grant from National Institutes of Health (MH60231), the Eric and Lila Nelson Chair in Neuropharmacology fund and the Medical Scientist Training Program. This work was also made possible, in part, through access to the Laser Microbeam and Medical Program (LAMMP) at the University of California, Irvine, under the National Institutes of Health Grant P41RR01192.
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The financial interests of Z.W., H.-P.N., and O.C. in NeoGene Technologies, Inc., have been reviewed by the Conflict of Interest Committee at the University of California, Irvine, and found to be acceptable by the state of California and/or the U.S. Government.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- PDZ
- PSD-95, Discs-large, ZO-1
- PrRP
- prolactin-releasing peptide
- AMPA
- α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
- PCR
- polymerase chain reaction
- PrRP-R
- prolactin-releasing peptide receptor
- HEK
- human embryonic kidney
- GRIP
- glutamate receptor interacting protein
- ABP
- α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding protein
- PBS
- phosphate-buffered saline
- PMA
- phorbol-12-myristate-13-acetate
- PICK1
- protein that interacts with C-kinase 1
- PKC
- protein kinase C
- DMSO
- dimethyl sulfoxide
- NHE
- Na+/H+ exchange
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- Received April 2, 2001.
- Accepted August 6, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
