TY - JOUR
T1 - Evidence for Spatial Proximity of Two Distinct Receptor Regions in the Substance P (SP)·Neurokinin-1 Receptor (NK-1R) Complex Obtained by Photolabeling the NK-1R with p-Benzoylphenylalanine3-Sp
AU - Bremer, Andrew A.
AU - Leeman, Susan E.
AU - Boyd, Norman D.
PY - 2001/6/22
Y1 - 2001/6/22
N2 - Substance P (SP) belongs to the tachykinin family of bioactive peptides and exerts its many biological effects through functional interaction with its cell-surface, G protein-coupled neurokinin-1 receptor (NK-1R). Previous studies from our laboratory have shown that 125I-Bolton-Hunter reagent-labeled p-benzoylphenylalanine8-SP (Bpa8SP) covalently attaches to Met181, whereas 125I-Bolton-Hunter reagent-labeled Bpa4SP covalently attaches to Met174, both of which are located on the second extracellular loop (EC2) of the NK-1R. In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue 125I-[D-Tyr0]Bpa3SP covalently labels residues in two distinct extracellular regions of the NK-1R. One site of 125I-[D-Tyr0]Bpa3SP photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of 125I-[D-Tyr0]Bpa3SP photoinsertion is located on the extracellular N terminus within a segment of the receptor extending from residues 11 to 21, a sequence that contains both potential sites for N-linked glycosylation. Since competition binding data presented in this study do not suggest the existence of multiple peptide·NK-1R complexes, it is reasonable to assume that the receptor sequences within EC2 and N terminus identified by peptide mapping are in close proximity in the equilibrium complex.
AB - Substance P (SP) belongs to the tachykinin family of bioactive peptides and exerts its many biological effects through functional interaction with its cell-surface, G protein-coupled neurokinin-1 receptor (NK-1R). Previous studies from our laboratory have shown that 125I-Bolton-Hunter reagent-labeled p-benzoylphenylalanine8-SP (Bpa8SP) covalently attaches to Met181, whereas 125I-Bolton-Hunter reagent-labeled Bpa4SP covalently attaches to Met174, both of which are located on the second extracellular loop (EC2) of the NK-1R. In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue 125I-[D-Tyr0]Bpa3SP covalently labels residues in two distinct extracellular regions of the NK-1R. One site of 125I-[D-Tyr0]Bpa3SP photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of 125I-[D-Tyr0]Bpa3SP photoinsertion is located on the extracellular N terminus within a segment of the receptor extending from residues 11 to 21, a sequence that contains both potential sites for N-linked glycosylation. Since competition binding data presented in this study do not suggest the existence of multiple peptide·NK-1R complexes, it is reasonable to assume that the receptor sequences within EC2 and N terminus identified by peptide mapping are in close proximity in the equilibrium complex.
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U2 - 10.1074/jbc.M100824200
DO - 10.1074/jbc.M100824200
M3 - Article
C2 - 11294866
AN - SCOPUS:0035933727
VL - 276
SP - 22857
EP - 22861
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 25
ER -