The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling.
A yeast two-hybrid screen performed to identify binding partners of the CaR (calcium-sensing receptor) intracellular tail identified the adaptor protein 14-3-3θ as a novel binding partner that bound to the proximal membrane region important for CaR expression and signalling. The 14-3-3θ protein directly interacted with the CaR tail in pull-down studies and FLAG-tagged CaR co-immunoprecipitated with EGFP (enhanced green fluorescent protein)-tagged 14-3-3θ when co-expressed in HEK (human embryonic kidney)-293 or COS-1 cells. The interaction between the CaR and 14-3-3θ did not require a putative binding site in the membrane-proximal region of the CaR tail and was independent of PKC (protein kinase C) phosphorylation. Confocal microscopy demonstrated co-localization of the CaR and EGFP-14-3-3θ in the ER (endoplasmic reticulum) of HEK-293 cells that stably expressed the CaR (HEK-293/CaR cells), but 14-3-3θ overexpression had no effect on membrane expression of the CaR. Overexpression of 14-3-3θ in HEK-293/CaR cells attenuated CaR-mediated Rho signalling, but had no effect on ERK (extracellular-signal-regulated kinase) 1/2 signalling. Another isoform identified from the library, 14-3-3ζ, exhibited similar behaviour to that of 14-3-3θ with respect to CaR tail binding, cellular co-localization and impact on receptor-mediated signalling. However, unlike 14-3-3θ, this isoform, when overexpressed, significantly reduced CaR plasma membrane expression. Results indicate that 14-3-3 proteins mediate CaR-dependent Rho signalling and may modulate the plasma membrane expression of the CaR.
‡Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, WA 6009, Australia.
Ajanthy
A
Arulpragasam
Aaron L
AL
Magno
Evan
E
Ingley
Suzanne J
SJ
Brown
Arthur D
AD
Conigrave
Thomas
T
Ratajczak
Bryan K
BK
Ward
D048948:14-3-3 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D011485:Protein Binding / Q000235:genetics; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology; D044169:Receptors, Calcium-Sensing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D014162:Transfection; D054460:rho-Associated Kinases / Q000378:metabolism*
D048948:14-3-3 Proteins; D044169:Receptors, Calcium-Sensing; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D054460:rho-Associated Kinases