Human Ca(2+) receptor extracellular domain. Analysis of function of lobe I loop deletion mutants.
The G protein-coupled Ca(2+) receptor (CaR) possesses an approximately 600-residue extracellular domain involved in ligand binding and receptor activation. Based on an alignment of the amino acid sequence of the CaR with that of bacterial periplasmic-binding proteins, the first approximately 530 residues of the extracellular domain are believed to form a domain resembling a bilobed Venus's flytrap (VFT). Four insertions in the CaR sequence that do not align with those of bacterial periplasmic-binding proteins correspond to four loops within lobe I of the VFT. We constructed a series of deletion mutants of these four loops and tested their ability to form fully processed CaR as well as their ability to be activated by Ca(2+). As many as 21 residues (365) of loop III could be deleted without impairing receptor expression or activation. Deletion of portions of either loops I (50) or IV (438) did not impair receptor expression but significantly reduced Ca(2+) activation. Deletion of the entire loop II (117) abolished receptor expression and function, but the replacement of even a single residue within this deletion mutant led to expression of a monomeric form of the receptor showing increased Ca(2+) sensitivity but reduced maximal activation. Our results reveal that certain residues within loops I and IV are dispensable in formation of the VFT domain but are critical for Ca(2+) activation of the receptor. In contrast, the residues in loop II are critical for maintaining the inactive state of the CaR. We discuss these results in light of the recently defined crystal structure of the homologous domain of the type 1 metabotropic glutamate receptor.
Reyes-Cruz
G
G
Molecular Pathophysiology Section, NIDCD, National Institutes of Health, Bethesda, Maryland 20892, USA. guadeluper@intra.niddk.nih.gov
Hu
J
J
Goldsmith
P K
PK
Steinbach
P J
PJ
Spiegel
A M
AM
D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017384:Sequence Deletion
D002135:Calcium-Binding Proteins