Cryptic initiation at the human D4 receptor reveals a functional role for the amino terminus.
It was found that deletion of the initiator methionine of the D4 receptor results in the use of a cryptic initiation site in the putative first transmembrane region. We made use of this observation to investigate the role of the amino terminus of the D4 receptor. In vitro transcription and translation of D4.4 and a D4.4 deleted for the initiation codon (D4.4 delta NH2) resulted in the formation of protein products with a molecular mass of about 44 and 40.5 kDa, respectively. The molecular mass of 40.5 kDa suggests initiation in the putative first transmembrane region. Transient expression of various deletion mutants indicated that this receptor form can be expressed at up to 70% of the D4.4 control levels and provided support for the existence for an alternative translation initiation site in the first transmembrane domain, most likely at nucleotide +112 (the initiator methionine codon is designated as +1). The D4.4 delta NH2 mutant was stably expressed in CHO cells. Pharmacological analysis demonstrated no major differences in antagonist binding with the regular D4.4 receptor, while dopamine and quinpirole binding affinities were about 5-fold decreased. The half-maximal level (EC50) for blocking forskolin-stimulated cAMP levels by dopamine was about 10-fold lower as compared to D4.4. Furthermore, the functional efficacy is decreased by about 40%. These data suggest that the amino-terminal domain is not essential for proper expression, but does interfere with the functional activity of the receptor, possibly through stabilization of the active state. To our knowledge this is the first demonstration that the amino terminus of a dopamine receptor is involved in signal transduction.
Schoots
O
O
Department of Psychiatry, Clarke Institute of Psychiatry, Toronto, Ontario, Canada.
Sanyal
S
S
Guan
H C
HC
Jovanovic
V
V
Van Tol
H H
HH
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D065127:Dopamine D2 Receptor Antagonists; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000502:physiology; D050638:Receptors, Dopamine D4; D011994:Recombinant Proteins; D017384:Sequence Deletion; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection
C493599:DRD4 protein, human; D065127:Dopamine D2 Receptor Antagonists; D008562:Membrane Glycoproteins; D017448:Receptors, Dopamine D2; D011994:Recombinant Proteins; D050638:Receptors, Dopamine D4