Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling.
OBJECTIVE
We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine.


METHODS
The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy.


RESULTS
The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle.


CONCLUSIONS
Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition.
Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol, UK.
S J
SJ
Mundell
A-L
AL
Matharu
S
S
Nisar
T M
TM
Palmer
J L
JL
Benovic
E
E
Kelly
D020816:Amino Acid Motifs; D000818:Animals; D019393:Arrestin / Q000378:metabolism*; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D034281:Dynamins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005973:Glutamine / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010649:Phenylalanine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D043704:Receptor, Adenosine A2B / Q000378:metabolism; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D017384:Sequence Deletion
D019393:Arrestin; D002966:Clathrin; D043704:Receptor, Adenosine A2B; D018047:Receptors, Purinergic P1; D005973:Glutamine; D010649:Phenylalanine; D034281:Dynamins; D007930:Leucine