We continued our investigations of the family 3 GPCRs with a study of the role of the linker sequences of the human calcium sensing receptor (hCaR) and the metabotropic glutamate receptor 1 (mGluR1). These studies were designed to reveal the role(s) played by this region of the family3 receptors in transmitting the ligand binding signal from the autonomously folding extracellular domain (ECD) to the transmembrane helix bundle (7TM) for G-protein regulation. Our findings are that disruption of this part of the hCaR structure in most cases leads to misfolded GPCR. Constructs in which a nine amino acid stretch of sequence within the 14 AA linker region was exchanged between the hCaR and mGluR1 yielded nearly wild-type function for both receptor types, highlighting the significant conservation of function of this region. Further, addition of glycine-rich, random coiled sequence, or replacement of this 9 AA with the random coil sequence resulted in misfolded receptors. However, a scanning alanine mutagenesis of the highly conserved residues within the linker sequence revealed 3 positions in which the mutations were tolerated and moderately activating of the hCaR and a fourth in which mutations produced significant misfolding and uncoupling of the ligand regulation through the ECD. These data complement our previous work suggesting essential contacts between the ECD, linker and the extracellular loops 2 and 3 of the 7TM domain. They also indicate that the ECD regulation of the 7TM domain involves changes in linker sequence contact(s) with the 7TM extracellular loop sequence(s) rather than a rotation about the 1st transmembrane domain transmitted by the linker. To complement our examination of the signaling properties of the 7TM cores of the family 3 GPCRs (hCaR and hT1Rs), we have constructed expression vectors for the ECD portions of the hCaR, mGluR1 and hT1Rs. Attempts to express these in several e.coli strains have uniformly failed, despite co-transformation of the expression hosts with identified bacterial chaperones and thioreductase. We are now constructing baculoviral vectors for this project, and we have initiated an examination of the cellular processing of family c GPCRs in mammalian cells. Our initial findings indicate that the 7TM cores and the ECD constructs utilize alternate sorting pathways within HEK293 cells which can be distinguished by COPII vesicle transport from ER to Golgi using Sar1 and Rab1 GTPases. The wild-type structures for hCaR and mGluR1 appear to utilize both sorting pathways. Surprisingly, the standard ER-export signal sequence does not appear to play a role in this sorting, and we are currently examining the molecular component(s) linking the ECD structure to Sar1/Rab1 recognition.