|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 26, 19955-19963, June 30, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Endocrine Research Unit, Department of Veteran Affairs
Medical Center, Department of Medicine, University of California,
San Francisco, California 94121
To determine the role of amino acids in the
second and third intracellular (IC) loops of the
Ca2+-sensing receptor (CaR) in phospholipase C (PLC)
activation, we mutated residues in these loops either singly or in
tandem to Ala and assessed PLC activity by measuring high extracellular [Ca2+] ([Ca2+]o)-induced inositol
phosphate accumulation and protein expression by immunoblotting and
immunocytochemistry in human embryonic kidney 293 cells. Two CaR
constructs in the second IC loop, F707A CaR and to a lesser extent
L704A CaR, demonstrated reduced activation of PLC, despite levels of
protein expression comparable with the wild-type (wt) CaR. Substitution
of Tyr or His for Phe-707, but not Leu, Val, Glu, or Trp, partially
restored the ability of high [Ca2+]o to activate
PLC. Eight residues in the third IC loop were involved in PLC
signaling. The responses to high [Ca2+]o in cells
expressing CaRs with Ala substitutions at these sites were <35% of
the wt CaR. The L798A, F802A, and E804A CaRs were dramatically impaired
in their responses to [Ca2+]o even up to 30 mM. Substitutions of Leu-798 with other hydrophobic
residues (Ile, Val, or Phe), but not with acidic, basic, or polar
residues, produced reduced responses compared with wt. Phe-802 could be
replaced with either Tyr or Trp with partial retention of the ability
to activate PLC. Glu-804 could only be substituted with Asp or Gln and
maintain its signaling capacity. Cell surface expression of the CaRs
mutated at Leu-798 and Phe-802 appeared normal compared with wt CaR.
Cell surface CaR expression was, however, reduced substantially in
cells expressing several mutants at position Glu-804 by confocal
microscopy. These studies strongly implicate specific hydrophobic and
acidic residues in the second and third IC loops of the parathyroid CaR
(and potentially larger stretches of the third loop) in mediating
efficient high [Ca2+]o-induced PLC activation and
or CaR expression.
Amino Acids in the Second and Third Intracellular Loops of the
Parathyroid Ca2+-sensing Receptor Mediate Efficient
Coupling to Phospholipase C*
*
This work was supported by Department of Veterans Affairs
Merit Review and National Institutes of Health Grants DK 43400 and DK
55846.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Endocrine Research
Unit, 111N, San Francisco VA Medical Center, 4150 Clement St., San
Francisco, CA 94121. Tel.: 415-750-2089; Fax: 415-750-6929; E-mail:
dolores@itsa.ucsf.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  H. I. Abdullah, P. L. Pedraza, J. C. McGiff, and N. R. Ferreri CaR activation increases TNF production by mTAL cells via a Gi-dependent mechanism Am J Physiol Renal Physiol, February 1, 2008; 294(2): F345 - F354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. H. Nissen, S. E. Christensen, L. Heickendorff, K. Brixen, and L. Mosekilde Molecular Genetic Analysis of the Calcium Sensing Receptor Gene in Patients Clinically Suspected to Have Familial Hypocalciuric Hypercalcemia: Phenotypic Variation and Mutation Spectrum in a Danish Population J. Clin. Endocrinol. Metab., November 1, 2007; 92(11): 4373 - 4379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Cheng, C. Tu, L. Rodriguez, T.-H. Chen, M. M. Dvorak, M. Margeta, M. Gassmann, B. Bettler, D. Shoback, and W. Chang Type B {gamma}-Aminobutyric Acid Receptors Modulate the Function of the Extracellular Ca2+-Sensing Receptor and Cell Differentiation in Murine Growth Plate Chondrocytes Endocrinology, October 1, 2007; 148(10): 4984 - 4992. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Chang, C. Tu, Z. Cheng, L. Rodriguez, T.-H. Chen, M. Gassmann, B. Bettler, M. Margeta, L. Y. Jan, and D. Shoback Complex Formation with the Type B {gamma}-Aminobutyric Acid Receptor Affects the Expression and Signal Transduction of the Extracellular Calcium-sensing Receptor: STUDIES WITH HEK-293 CELLS AND NEURONS J. Biol. Chem., August 24, 2007; 282(34): 25030 - 25040. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. Reyes-Ibarra, A. Garcia-Regalado, I. Ramirez-Rangel, A. L. Esparza-Silva, M. Valadez-Sanchez, J. Vazquez-Prado, and G. Reyes-Cruz Calcium-Sensing Receptor Endocytosis Links Extracellular Calcium Signaling to Parathyroid Hormone-Related Peptide Secretion via a Rab11a-Dependent and AMSH-Sensitive Mechanism Mol. Endocrinol., June 1, 2007; 21(6): 1394 - 1407. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Binet, B. Duthey, J. Lecaillon, C. Vol, J. Quoyer, G. Labesse, J.-P. Pin, and L. Prezeau Common Structural Requirements for Heptahelical Domain Function in Class A and Class C G Protein-coupled Receptors J. Biol. Chem., April 20, 2007; 282(16): 12154 - 12163. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. D. Mittelman, G. N. Hendy, R. A. Fefferman, L. Canaff, I. Mosesova, D. E. C. Cole, L. Burkett, and M. E. Geffner A Hypocalcemic Child with a Novel Activating Mutation of the Calcium-Sensing Receptor Gene: Successful Treatment with Recombinant Human Parathyroid Hormone J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2474 - 2479. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Rodriguez, C. Tu, Z. Cheng, T.-H. Chen, D. Bikle, D. Shoback, and W. Chang Expression and Functional Assessment of an Alternatively Spliced Extracellular Ca2+-Sensing Receptor in Growth Plate Chondrocytes Endocrinology, December 1, 2005; 146(12): 5294 - 5303. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Rodriguez, Z. Cheng, T.-H. Chen, C. Tu, and W. Chang Extracellular Calcium and Parathyroid Hormone-Related Peptide Signaling Modulate the Pace of Growth Plate Chondrocyte Differentiation Endocrinology, November 1, 2005; 146(11): 4597 - 4608. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Bettler, K. Kaupmann, J. Mosbacher, and M. Gassmann Molecular Structure and Physiological Functions of GABAB Receptors Physiol Rev, July 1, 2004; 84(3): 835 - 867. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Chen and W. G. Goodman Role of the calcium-sensing receptor in parathyroid gland physiology Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1005 - F1011. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Havlickova, J. Blahos, I. Brabet, J. Liu, B. Hruskova, L. Prezeau, and J.-P. Pin The Second Intracellular Loop of Metabotropic Glutamate Receptors Recognizes C Termini of G-protein {alpha}-Subunits J. Biol. Chem., September 12, 2003; 278(37): 35063 - 35070. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Havlickova, L. Prezeau, B. Duthey, B. Bettler, J.-P. Pin, and J. Blahos The Intracellular Loops of the GB2 Subunit Are Crucial for G-Protein Coupling of the Heteromeric gamma -Aminobutyrate B Receptor Mol. Pharmacol., August 1, 2002; 62(2): 343 - 350. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Chang, C. Tu, S. Pratt, T.-H. Chen, and D. Shoback Extracellular Ca2+-Sensing Receptors Modulate Matrix Production and Mineralization in Chondrogenic RCJ3.1C5.18 Cells Endocrinology, April 1, 2002; 143(4): 1467 - 1474. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. D'Souza-Li, B. Yang, L. Canaff, M. Bai, D. A. Hanley, M. Bastepe, S. R. Salisbury, E. M. Brown, D. E. C. Cole, and G. N. Hendy Identification and Functional Characterization of Novel Calcium-Sensing Receptor Mutations in Familial Hypocalciuric Hypercalcemia and Autosomal Dominant Hypocalcemia J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1309 - 1318. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Duthey, S. Caudron, J. Perroy, B. Bettler, L. Fagni, J.-P. Pin, and L. Prezeau A Single Subunit (GB2) Is Required for G-protein Activation by the Heterodimeric GABAB Receptor J. Biol. Chem., January 25, 2002; 277(5): 3236 - 3241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Chang, S. Pratt, T.-H. Chen, L. Bourguignon, and D. Shoback Amino Acids in the Cytoplasmic C Terminus of the Parathyroid Ca2+-sensing Receptor Mediate Efficient Cell-surface Expression and Phospholipase C Activation J. Biol. Chem., November 16, 2001; 276(47): 44129 - 44136. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |