Family C receptors of the G-protein coupled receptor superfamily include metabotropic ?-aminobutyric acid (GABA)-B receptors (GABA-B-Rs), metabotropic glutamate receptors (mGluRs), and Ca2+-sensing receptors (CaRs). These receptors function in dimeric or multimeric forms which maintain the proper conformations for ligand binding, trafficking, and coupling to downstream effectors. A functional GABA-B-R consists of a heterodimer between the GABA-B-R1 and R2, two distinct gene products. CaRs function as homodimers in target cells (i.e., parathyroid and kidney), and they also can heterodimerize with mGluRs in the brain. We recently found that CaRs can also form heteromeric complexes with GABA-B-R 1s in HEK-293 cells transfected with both receptor cDNAs. Co-expressing GABA-B-R1s along with CaRs suppressed CaR protein levels and the responsiveness of phospholipase C activation in the cells to changes in the extracellular [Ca2+] ([Ca2+]e), thus supporting the idea that GABA-B-R1s alter CaR expression and function via a heteromeric receptor complex. Whether such co-associations occur in the target cells in vivo has received little attention. Both CaRs and GABA-B-Rs are present in many tissues - brain, kidney, parathyroid, intestine, bone and cartilage. In cultured mouse growth plate chondrocytes (mGPCs), knocking out the GABA-B-R1 genes dramatically increased the level of CaR protein and signaling responses to high [Ca2+]e, supporting a role for GABA-B-R1s in regulating the expression and function of the CaRs in mGPCs. Our hypothesis is that GABA-B-R1s modulate the expression and signal transduction of CaRs by the formation of heteromeric complexes between the two molecules and that this interaction impacts on the signaling responses and function of target cells. To test this hypothesis, we propose the following aims.
AIM 1 : To determine whether CaRs and GABA-B-R1s interact to modulate CaR expression and signal transduction. We will address this by (a) co-expressing GABA-B-R1s with CaRs in HEK-293 cells and assessing whether their association affects the synthesis and or degradation of CaR protein and alters the signal transduction properties of CaRs; (b) testing the effects of blocking GABA-B-R1 expression in mGPCs on the synthesis and or degradation of CaR protein and on CaR-mediated signal transduction; (c) ascertaining whether GABA-B-R1s co-associate with CaRs in mGPCs; and (d) examining the effects of blocking GABA-B-R1 expression on the proliferation, apoptosis, and expression of markers of differentiation -- processes responsive to changes in the [Ca2+]e in mGPCs.
AIM 2 : To determine the impact of the GABA- B-R1 in cartilage in vivo by examining the effect of cartilage-specific knockout of the GABA-B-R1 genes on CaR expression, morphology, cell proliferation, apoptosis, and the expression of markers of differentiation in growth plate cartilage in mice. Completion of this work will provide insights into how CaRs and GABA-B-R1s interact in a physiological context and how such an interaction influences cartilage development. CaRs control mineral balance in the body, and GABA-B-Rs are essential to nerve cell function. The growth plate provides the cells and proteins that initiate new bone formation, which ultimately determines our height and body size. This work can open up future studies targeted to developing therapies to modulate the function of CaRs, GABA-B-Rs, or the heteromeric complexes of these two receptors. ? ? ?
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