Synthesis and processing of D2 dopamine receptors.
Dopamine receptors belong to a superfamily of neurotransmitter receptors that are functionally coupled to guanine nucleotide binding proteins. In this study, we have used Chinese hamster ovary (CHO) cells stably transfected with the rat D2L receptor, in conjunction with specific anti-peptide antibodies that we have developed, in order to visualize this protein and the course of its synthesis. The newly synthesized receptor exists as a 45-kDa protein which undergoes further processing to a 75-kDa glycosylated receptor in the CHO cells. In pulse-chase experiments it was noticed that a 35-kDa precursor was present which disappeared after 30 min. In order to determine whether this 35-kDa protein represents an unprocessed form of the receptor, we have employed an in vitro translation system with cDNA constructs coding for both the murine D2 and D3 dopamine receptor isoforms. In the absence of processing, the D2 and D3 receptors have an apparent molecular mass of 35 kDa. The translated proteins were shown to be the full length receptors by immunoprecipitation with various anti-peptide antibodies and by the demonstration that they can undergo glycosylation to apparent molecular masses of approximately 45 kDa in an in vitro system.
David
C
C
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Fishburn
C S
CS
Monsma
F J
FJ
Sibley
D R
DR
Fuchs
S
S
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D006031:Glycosylation; D007163:Immunosorbent Techniques; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis; D051381:Rats; D017448:Receptors, Dopamine D2 / Q000096:biosynthesis* / Q000235:genetics; D013134:Spiperone / Q000031:analogs & derivatives; D014162:Transfection
D000345:Affinity Labels; D001386:Azides; D011498:Protein Precursors; D017448:Receptors, Dopamine D2; D013134:Spiperone; D004247:DNA; C044567:N-(4-azido-3-iodophenethyl)spiperone