Differential regulation of muscarinic M1 and M3 receptors by a putative phosphorylation domain.

A motif consisting of several serine residues flanked N-terminally by acidic residues occurs in the third intracellular loop of both muscarinic M1 and M3 receptors (287SerLeuThrSerSer291 and 349SerAlaSerSer352, respectively). We examined the role of these domains in modulating agonist-induced desensitization and receptor trafficking, and for the muscarinic M3 receptor, we assessed the contribution of phosphorylation to receptor regulation. Mutation of the above residues did not affect desensitization of phosphoinositide hydrolysis signaling for either the muscarinic M1 or M3 receptor and did not alter the agonist-induced phosphorylation state of the muscarinic M3 receptor. Mutation of this domain (349SerAlaSerSer352/349AlaAlaAlaAla352) in the muscarinic M3 receptor completely abrogated receptor internalization and subsequently, down-regulation. Mutation of the analogous domain (287SerLeuThrSerSer291/287AlaLeuAlaAlaAla291) in the muscarinic M1 receptor had no obvious effect on internalization, but led to a more rapid down-regulation. Thus, these serine-rich regions are not required for receptor desensitization, but are differentially involved in receptor trafficking for the muscarinic M1 and M3 receptors.

Chemokine receptor CXCR4 expression in endothelium.

The expression of chemokine receptor and viral coreceptor CXCR4 is reported in cultured endothelial cells and in arterial endothelium. A 1.9 kb transcript was cloned from cultured bovine aortic (BAEC) and human umbilical vein endothelial cells (HUVEC). CXCR4 mRNA was expressed at high levels in BAEC and HUVEC but was not expressed by cultured bovine arterial smooth muscle cells (BASM) or human umbilical vein smooth muscle cells (HUVSM). Western blotting with polyclonal antibodies demonstrated an approximate 46KD protein in endothelial cells only. In situ hybridization and immunocytochemistry (anti-CXCR4 monoclonal antibody 12G5) revealed both transcript and protein expression in cultured endothelial cells, and in the endothelium of normal aorta but not in aortic smooth muscle. The ligand for CXCR4, stromal cell derived factor 1 (SDF-1) stimulated mobilization of intracellular calcium at a moderate level (37% of the peak response to thrombin), confirming the expression of functional receptor at the endothelial surface. The involvement of CXCR4 in chemokine signaling, chemoattraction (through SDF-1), and its potential viral coreceptor activity suggest a multifunctional role in vascular homeostasis and pathophysiology.

Residues specifically involved in down-regulation but not internalization of the m1 muscarinic acetylcholine receptor.

Human m1 muscarinic acetylcholine receptor mutants were screened to determine receptor domains and cellular pathways relevant to down-regulation. Mutations in the second intracellular loop and the junctions of the third intracellular loop of the receptor, where a role for receptor activation or internalization had been previously demonstrated in HEK293 cells, were selected for this study. To assess receptor down-regulation, the m1 receptor mutants were transfected into Chinese hamster ovary cells. Because receptor internalization is expected to precede down-regulation, mutants displaying intact internalization were selected to permit interpretation of mutational effects on down-regulation alone. Four mutations were identified that specifically impaired down-regulation without altering receptor internalization: V127A, I211A, E360A, and K362A. The results define new receptor domains in the second intracellular loop and the junctions of the third intracellular loop that are involved in down-regulation. These same four mutants were also defective in signaling via the phospholipase C and the adenylyl cyclase pathways and in G protein activation, as measured by [35S]GTP gamma S binding. However, the level of second messenger stimulation correlated poorly with the extent of down-regulation. In summary, several mutations of the m1 receptor selectively affect down-regulation, demonstrating that internalization and down-regulation represent distinct events driven by different cellular mechanisms.

Activating and inactivating mutations in N- and C-terminal i3 loop junctions of muscarinic acetylcholine Hm1 receptors.

The N- and C-terminal junctions of the third intracellular loop (i3) of G protein-coupled receptors play a role in the coupling process. We had previously constructed two triple point alanine mutants of the i3 junction of the muscarinic Hm1 receptor, W209A/I211A/Y212A and E360A/K362A/T366A, which are defective in mediating carbachol stimulation of phosphatidylinositol (PI) turnover (Moro, O., Lameh, J., Högger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 22273-22276). Each of the corresponding six single point mutations were constructed to determine residues crucial to receptor coupling. Mutants W209A and T366A were similar to or only slightly less effective than wild type Hm1 in stimulating PI turnover. In the N-terminal junction, I211A and Y212A were defective in coupling, and I211A was even more defective than the corresponding triple mutant. Therefore, the triple mutation compensated at least partially for the effect of these two single point mutations. In the C-terminal i3 loop junction, mutant K362A was again more strongly defective than the corresponding triple mutant. In contrast, mutation E360A was found to be activating, leading to elevated PI turnover in the absence of agonist and sensitization toward carbachol activation. Activating mutations in the C-terminal i3 loop junction have been reported previously for the adrenergic receptors, but E360A represents the first muscarinic receptor with substantial basal activity. The effects of the single point mutations observed in this study were not readily predictable from similar mutations from closely related G protein-coupled receptors despite sequence conservation in the i3 loop junctions. Our results caution against defining precise coupling domains in these regions by mutagenesis results.

Overlapping multi-site domains of the muscarinic cholinergic Hm1 receptor involved in signal transduction and sequestration.

Alanine mutagenesis scanning of the intracellular portion of the human muscarinic cholinergic Hm1 receptor was performed to identify domains mediating agonist induced receptor sequestration. Using these multiple alanine point mutants of Hm1, we had previously identified several receptor domains in the intracellular loops i1-3 that play a role in coupling to phosphatidyl inositol turnover, most notably, a lipophilic residue, Leu-131, in the conserved i2 loop domain DRYXXVXXPL (Moro, O., Lameh, J., Hogger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 6862-6865). We now demonstrate that alanine substitutions in three of these domains, i.e. middle of the i2 loop and both junctions of the i3 loop, also result in defective sequestration (loss of surface receptor sites accessible to a polar tracer) in transfected human kidney U293 cells. The i2 loop was studied further by single point mutations. The strongest impairment of sequestration occurred with mutant L131A which was also highly defective in phosphatidyl inositol (PI) coupling. Substitution of Leu-131 with several distinct amino acids indicated that a bulky lipophilic residue is required for sequestration in this position, as shown for coupling to PI turnover. Further, the double point mutation, V127A/L131A, almost completely suppressed both sequestration and coupling of Hm1. In the beta 2 adrenoceptor, alanine substitution of the i2 residue Phe-139, equivalent to Leu-131 in Hm1, also resulted in impaired coupling to adenylyl cyclase and sequestration, indicating a general role for this conserved i2 loop residue in both processes. The combined results show that the multi-site domain involved in signal transduction of Hm1 is similar to and overlaps with that involved in sequestration. However, three Hm1 mutants that were moderately deficient in stimulating PI turnover displayed normal sequestration, suggesting distinct mechanisms. We propose that cellular mediators of receptor sequestration are structurally similar or identical to the heterotrimeric G proteins.

Mutational analysis of third cytoplasmic loop domains in G-protein coupling of the HM1 muscarinic receptor.

We measured dose-response curves for carbachol stimulation of phosphatidyl inositol (PI) turnover with mutants of the Hm1 muscarinic cholinergic receptor having various deletions from amino acids 219 to 358 of the large third intracellular (i3) loop (208 to 366). These deletions had only small or no effects on the ability of Hm1 transfected into HEK 293 cells to stimulate PI turnover. This result indicates that only small regions of 9 to 11 amino acids adjacent to trans-membrane domains (TMDs) 5 and 6 can be directly involved in G protein coupling. Point mutations were constructed to test the role of charged amino acids in these junctions. A triple point mutation of Hm1 (E214 A/ E216K/ E221 K), which mimics the charge distribution in Hm2 (negatively coupled to cAMP) over the first 14 amino acids of i3, and a double point mutation in the N terminal junction, K359A/K361A, both failed to affect carbachol stimulated PI turnover. Therefore, charge distribution in the loop junctions appears to play a minor role in G protein coupling of Hm1 in HEK 293 cells.