Functional interactions between the extracellular domain and the seven-transmembrane domain in Ca2+ receptor activation.
We studied the activity of mutants involving the aminoterminal extracellular, seven-transmembrane (7TM) and carboxy-terminal tail domains of the human Ca2+ receptor to gain insight into the functional interactions between these domains during receptor activation. Missense mutations of highly conserved residues, D190 and E297, in the extracellular domain (ECD), and a mutation within part of the proximal carboxyterminal tail, A877-880E, resulted in receptors with severely reduced response to Ca2+ despite adequate cell surface expression. Coexpression of either D190A or E297K mutants with A877-880E led to significant reconstitution of function. No such reconstitution occurred when D190A or E297K mutants were coexpressed with a truncation mutant possessing an intact amino-terminal extracellular and first transmembrane domain, despite evidence for heterodimerization and cell surface expression of the respective mutant receptors. In addition, no reconstitution of function was observed when D190A was coexpressed with a deletion Ca2+ receptor mutant lacking only a cysteine-rich region located in the ECD of the Ca2+ receptor (Ca-//-Ca). Moreover, coexpression of this Ca-//-Ca with A877-880E did not recover function. The results show that Ca2+ receptor extracellular and 7TM domains are discrete entities that can communicate within the context of a heterodimer composed of complementary mutant receptors. Two intact 7TM domains and two intact cysteine-rich regions appear to be required for such communication to occur. The results are discussed in the context of a speculative model of receptor structure and function.
Hauache
O M
OM
Metabolic Diseases Branch, NIDDK, NIH, Bethesda, MD, USA.
Hu
J
J
Ray
K
K
Spiegel
A M
AM
D000595:Amino Acid Sequence; D019921:Biotinylation; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D015870:Gene Expression; D006801:Humans; D006868:Hydrolysis; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D002135:Calcium-Binding Proteins; D010716:Phosphatidylinositols; D002118:Calcium