Functional expression of olfactory-adrenergic receptor chimeras and intracellular retention of heterologously expressed olfactory receptors.
Replacing the G-protein-coupling domains of the beta 2-adrenergic receptor with homologous domains of putative olfactory receptors produced chimeric receptors which were able to stimulate pigment dispersion in Xenopus melanophores, a G-protein-mediated pathway. A multiple replacement chimera containing the second, third and C-terminal cytoplasmic domains of receptor OR5 elevated cyclic adenosine 3':5'-monophosphate (cAMP) and suppressed production of inositol phosphates. Co-expression of G alpha olf did not alter the strength of response of this chimera. A novel rat olfactory receptor cDNA (U131) was isolated and sequenced. Expression of U131 and OR5 constructs containing an N-terminal epitope-tag or C-terminal fusion to green fluorescent protein occurred in an intracellular network but not in the plasma membrane of heterologous cells. Similarly treated beta 2-adrenergic receptors were functional and were observed in the plasma membrane and the intracellular network. These results demonstrate that the putative cytoplasmic domains of olfactory receptors are capable of functional interaction with heterologous G-proteins of the G alpha s subtype. Instead, the absence of these receptors from the plasma membrane of heterologous cells appears to explain our inability to determine if odorants can activate the olfactory receptor clones. We hypothesize that the olfactory receptors have requirements for maturation and targeting to the plasma membrane that are different from most other G-protein-coupled receptors.
McClintock
T S
TS
Department of Physiology, University of Kentucky, Lexington 40536-0084, USA. mcclint@pop.uky.edu
Landers
T M
TM
Gimelbrant
A A
AA
Fuller
L Z
LZ
Jackson
B A
BA
Jayawickreme
C K
CK
Lerner
M R
MR
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D004789:Enzyme Activation; D008547:Melanophores / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D011941:Receptors, Adrenergic / Q000096:biosynthesis*; D018035:Receptors, Odorant / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D018076:DNA, Complementary; D011941:Receptors, Adrenergic; D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins; D000262:Adenylyl Cyclases