Functional expression of heteromeric calcitonin gene-related peptide and adrenomedullin receptors in yeast.
The ability of G protein-coupled receptors (GPCRs) to form homo- and heteromeric complexes has important implications for the regulation of cellular events. A notable example of heteromer formation is the interaction of the calcitonin receptor-like receptor (CRLR) with different members of the receptor activity modifying protein (RAMP) family, which results in the formation of two different receptors, a calcitonin gene-related peptide (CGRP) receptor and an adrenomedullin receptor. To analyze the role of RAMPs in determining ligand specificity, we have co-expressed CRLR and RAMP proteins in the yeast Saccharomyces cerevisiae, which provides a null system to study the function of mammalian receptors. Co-expression of RAMP1 and CRLR reconstituted a CGRP receptor that was able to activate the pheromone-signaling pathway with pharmacological properties similar to those observed previously in mammalian cells. Co-expression of CRLR with RAMP2 or RAMP3 resulted in a response with the pharmacological properties of an adrenomedullin receptor. These data indicate that RAMPs are necessary and sufficient to determine ligand specificity of CRLR. Contrary to observations in mammalian cells, the glycosylation of CRLR was not affected by the presence of RAMPs in yeast, indicating that glycosylation of CRLR is not the prime determinant of ligand specificity. The first functional reconstitution of a heteromeric seven transmembrane receptor in yeast suggests this organism as a useful research tool to study the molecular nature of other heteromeric receptors.
Miret
Juan J
JJ
OSI Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591-6705, USA.
Rakhilina
Larissa
L
Silverman
Lauren
L
Oehlen
Bert
B
D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000096:biosynthesis* / Q000737:chemistry*; D058286:Calcitonin Receptor-Like Protein; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D015723:Gene Library; D006031:Glycosylation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018003:Receptors, Calcitonin / Q000096:biosynthesis / Q000737:chemistry; D018000:Receptors, Peptide / Q000096:biosynthesis* / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D015398:Signal Transduction
C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; C545502:RAMP1 protein, human; C545503:RAMP2 protein, human; C545504:RAMP3 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018003:Receptors, Calcitonin; D018000:Receptors, Peptide; D000242:Cyclic AMP; D015740:Calcitonin Gene-Related Peptide